Meticulous Control of the T3SS of Yersinia Is Essential for Full Virulence

Meticulous control of the T3SS of Yersinia is essential for full virulence

Ann-Catrin Björnfot

Department of Molecular Biology Umeå Centre for Microbial Research (UCMR) Laboratory for Molecular Infection Medicine Sweden (MIMS) Umeå University, Sweden Umeå 2011

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TABLE OF CONTENTS

TABLE OF CONTENTS i ABSTRACT iii ABBREVIATIONS iv PAPERS IN THIS THESIS v INTRODUCTION 1 1. Gram-negative bacteria 1 1.1 Enteropathogenic bacteria 1 1.2 Human pathogenic species of the genus Yersinia 2 1.2.1 Y. pestis has evolved from Y. pseudotuberculosis 2 1.2.2 Diseases and symptoms coupled to Yersinia - Plague and Gastroenteritis 3 1.2.3 Yersinia species and their intracellular nature 4 2. Secretion systems in gram-negative bacteria 4 2.1 Secretion across the bacterial cytoplasmic membrane 5 2.1.1 The general secretory system (Sec) 5 2.1.2 The signal recognition particle system (SRP) 5 2.1.3 Twin-arginine translocation (Tat) 6 2.2 Secretion across the outer membrane 6 2.2.1 Type I secretion (T1S) 7 2.2.2 Type II secretion (T2S) 7 2.2.3 Type IV secretion (T4S) 7 2.2.4 Type V secretion (T5S) 8 2.2.5 The chaperone/usher (CU) pathway 8 2.2.6 Type VI secretion (T6S) 9 2.3 The Type III secretion system (T3SS) 9 2.4 The flagellar T3SS 11 2.4.1 FliK and FlhB – The flagellar substrate specificity switch 12 2.5 The Yersinia T3SS 13 2.5.1 The type III secretion apparatus – Ysc proteins 13 2.5.2 Type III effector proteins 15 2.5.3 Translocator proteins of Yersinia 16 2.5.4 Chaperones of the Yersinia type III secretion system 18 2.5.5 Type III effector secretion signals 19 2.6 Regulation of the Yersinia T3SS 19 2.6.1 The role of temperature 19 2.6.2 The low calcium response (LCR) 20 2.6.3 Other Yersinia T3SS regulatory elements 20 2.6.4 Target cell contact dependent Yersinia T3SS regulation 21 2.7 Secretion control performed by YscU and YscP 22

i 2.7.1 YscU and the conserved NPTH motif 23 2.7.2 On the role of YscP 24 3. Bacterial stress response 25 3.1 Heat shock response 25 3.2 Protein aggregate disaggregation and protein folding 25 3.2.1 DnaK/DnaJ/GrpE 26 3.2.2 The ATPase ClpB 27 4. Antibiotics and resistance 28 4.1 Antibiotic action and resistance to antibiotics 28 4.2 Bacterial protein translation and the ribosome 29 4.3 The T3SS as a target of antimicrobial compounds 32 4.4 Virulence-associated genes (Vag) as potential targets for antimicrobial therapy 32 AIMS 34 RESULTS AND DISCUSSION 35 5. YscU and its autoproteolytic ability – On the connection to T3SS regulation (Paper I) 35 5.1 YscU is autoproteolytically cleaved in the NPTH motif 35 5.2 Disturbed autoproteolysis affects regulation of T3SS expression and secretion 35 5.3 Proper YscU function is needed for needle regulation 36 5.4 Motif mutants assemble functional T3SS machines 37 5.5 Proposed model of YscU action 37 6. DnaK/DnaJ are needed for proper T3SS function (Paper II) 39 6.1 DnaK/DnaJ are needed for a functional flagellar T3SS and Yop T3SS 39 6.2 The connection between the HSPs and T3SS regulation 40 6.3 Full-length YscU associates with the bacterial OM 40 6.4 DnaJ specifically interacts with defined YscU domains 41 6.5 Model of DnaK/DnaJ T3SS regulation contribution 41 7. VagH and the link to the T3SS and virulence (Paper III) 43 7.1 VagH has methyltransferase activity and targets RF1 and RF2 43 7.2 Microarray and proteomics - VagH in the scene of T3SS regulation 44 7.3 VagH is needed for virulence and macrophage survival 44 8. Further remarks 45 CONCLUSIONS 47 ACKNOWLEDGEMENTS 48 REFERENCES 51 PAPERS I-III

ii ABSTRACT

The type III secretion system (T3SS) of pathogenic Yersinia pseudotuberculosis is involved in virulence. The syringe-like secretion system spans both bacterial membranes and is responsible for the ability of Yersinia to transfer toxic proteins (Yop proteins) into the eukaryotic target cell. The T3SS is believed to have evolved from the flagellum and regulation of the T3SS is a complex event that involves a series of regulatory proteins, whereby two are YscP and YscU. In a regulatory model, called the substrate specificity switch, both proteins act together to ensure proper T3SS structure and function by regulating a stop in YscF needle protein export with a shift to Yop effector secretion. YscU undergoes autoproteolysis at a conserved motif consisting of amino acids Asparagine-Proline-Threonine-Histidine

(NPTH). Processing generates a C-terminal 10 kDa peptide, YscUCC. Processing is crucial for proper T3SS regulation and function both in vitro and in vivo. Full-length YscU does not support Yop secretion and after cleavage, YscUCC remains attached to the rest of YscU and acts as a negative block on T3S. Relief of this negative block is suggested to occur through displacement of YscUCC from the rest of YscU. Thorough control of many different cellular processes is brought by the heat shock proteins (HSPs) DnaK and DnaJ. Due to their multiple regulatory functions, mutations in the hsp-genes lead to pleiotropic effects. DnaK and DnaJ are essential for proper flagellum driven motion of bacteria, but more so; they ensure proper Yersinia T3SS function in vivo. Furthermore, DnaJ interacts with YscU and may be directly involved in T3SS regulation. Virulence of Yersinia is regulated on many levels. A previously identified virulence associated protein, VagH, is now characterized as an S-adenosyl-methionine dependent methyltransferase. The targets of the methylation activity of VagH are release factors 1 and 2 (RF1 and RF2), that are important for translation termination. The enzymatic activity of VagH is important for Yop secretion and a vagH mutant up-regulates a T3SS negative regulatory protein, YopD. Furthermore, a vagH mutant is avirulent in a mouse infection model, but is not affected in macrophage intracellular survival. The importance of VagH in vivo makes it a possible target for novel antimicrobial therapy.

Keywords

Yersinia, type III secretion, YscU, substrate specificity switch, heat shock proteins, VagH, methyltransferase, virulence

iii ABBREVIATIONS

OM Outer membrane IM Inner membrane LPS Lipopolysaccharide Sec General secretory system SRP Signal recognition particle system Tat Twin-arginine translocation T1SS Type I secretion system T2SS Type II secretion system T3SS Type III secretion system T4SS Type IV secretion system T5SS Type V secretion system T6SS Type VI secretion system T7SS Type VII secretion system AT Autotransporter TPS Two-partner secretion Oca Oligomeric coiled-coil adhesin Ysc Yersinia secretion protein Yop Yersinia outer protein Syc Specific Yop chaperone LCR Low calcium response CD Calcium dependent CI Calcium independent TS Temperature sensitive wt Wild type Hsp Heat shock protein MDR Multi drug resistant VRSA Vancomycin resistant Staphylococcus aureus VRE Vancomycin resistant enterococci SD Shine-Dalgarno RNA Ribonucleic acid. rRNA-Ribosomal. mRNA-Messenger. tRNA-Transfer. IF Initiation factor RF Release factor SAM S-adenosyl-methionine MTase Methyltransferase NEF Nucleotide exchange factor HTS High-throughput screening Vag Virulence associated gene

iv PAPERS IN THIS THESIS

This thesis is based on the following publications and manuscripts referred to by their roman numerals (I - III)

I. Ann-Catrin Björnfot, Moa Lavander, Åke Forsberg, Hans Wolf-Watz. Autoproteolysis of YscU of Yersinia pseudotuberculosis is important for regulation of expression and secretion of Yop proteins. Journal of Bacteriology (2009) 191(13): 4259-67. Reprinted with permission from the publisher.

II. Ann-Catrin Björnfot, Frédéric H. Login, Tomas Edgren, Roland Nordfelth, Hans Wolf-Watz. Involvement of the heat shock proteins DnaK/DnaJ in Yersinia T3S. Manuscript.

III. Sara Garbom, Martina Olofsson, Ann-Catrin Björnfot, Manoj Kumar Srivastava, Victoria L. Robinson, Petra C.F. Oyston, Richard W. Titball, Hans Wolf-Watz. Phenotypic characterization of a virulence-associated protein, VagH, of Yersinia pseudotuberculosis reveals a tight link between VagH and the type III secretion system. Microbiology (2007) 153(Pt 5):1464-73. Reprinted with permission from the publisher.

INTRODUCTION

1. Gram-negative bacteria

By microscopic use, bacteria were first observed in the late 17th century. Within the group bacteria a large variety is found. Bacteria differ in shape and size and their habitats vary greatly. Common features that are shared by all bacteria are DNA replication, transcription and protein translation. The nucleus is not membrane-bound and the DNA is normally found as a circular chromosome in the cytoplasm. The diversity of bacterial species can be described by naming and grouping organisms based on resemblances. One way to classify bacteria is to divide them into gram positives and gram negatives, a method developed by the Danish Hans Christian Gram in the late 19th century. In this grouping bacteria are distinguished by their membrane characteristics (Figure 1). The Gram-positive cell wall consists of a thick layer of peptidoglycan while the gram-negative cell wall is constituted by an inner (IM) and an outer membrane (OM) separated by a periplasmic space lined with a thin layer of peptidoglycan. The external features of bacteria differ from species to species and also vary within species. Surface exposed proteins and structures are important for bacterial infection and invasion and also aid in bacterial protection against the host immune system response.

Figure 1. Characterization of bacteria depending on their membrane characteristics. Gram-negative bacteria (G-) have an inner membrane (IM) and an outer membrane (OM). A periplasmic space containing peptidoglycan separates the two membranes. Gram-positive (G+) bacteria have an IM and their outmost is made of a thick layer of peptidoglycan.

1.1 Enteropathogenic bacteria

The bacterial family Enterobacteriaceae includes many of the more familiar pathogens, such as Salmonella, Escherichia coli and Yersinia. Bacteria of the family are characterized as gram-negative, rod-shaped, facultative anaerobes. Enterobacteriaceae members do not form spores and most often they are flagellated. In the gut many members of the family are a part of the normal flora whereas others are found more often in soil or water. The gut is a complex symbiotic environment where normal gut microbiota benefits

from the environment supplied by the host and the host benefits from the nutritional support and the contribution in immune system development by the bacterium. Upon infection of the gut flora with enteropathogenic bacteria, this symbiotic environment may be disturbed and symptoms such as for instance diarrhea can appear. Unique to the pathogenic class of bacteria is their capacity to evoke disease by so called virulence factors. Virulence factors aid in bacterial adherence to host cells, invasion of the cell and survival inside the cell. The distribution of virulence factors vary among bacterial species and the factors may be located on the bacterial surface, be released to the external milieu, or translocated over the bacterial membranes directly into the host cell cytosol. The transfer of proteins over the bacterial membranes occurs via specific secretion systems. These systems are inserted in either bacterial membrane or may span both membranes. The following sections will cover the enteropathogen Yersinia and diseases coupled to the bacterium. Yersinia utilizes a specific secretion system to evoke disease. This secretion system will be discussed in detail later (See sections 2.3-2.7.2).

1.2 Human pathogenic species of the genus Yersinia

The most known of all Yersinia species is Yersinia pestis, the causative agent of plague. The genus Yersinia consists of 11 species, whereby three are human pathogens; Yersinia pseudotuberculosis, Yersinia enterocolitica and Yersinia pestis. The eight less studies species (Y. aldovae, Y. frederiksenii, Y. bercovieri, Y. kristensenii, Y. rohdei, Y. ruckeri, and Y. intermedia, Y. mollaretii) are considered nonpathogenic and lack classical virulence markers specific for Yersinia. The group of eight is often referred to as Yersinia enterocolitica-like due to earlier grouping made from lack of information of the group members. (335)

1.2.1 Y. pestis has evolved from Y. pseudotuberculosis

Evolutionary studies of Yersinia ancestry map Yersinia pestis as an emerged clone of Yersinia pseudotuberculosis. The division occurred 1.500-20000 years ago. (4) Depending on the ability to reduce nitrate and ferment glycerol, Y. pestis is divided into three biovars: Orientalis, Medievalis and Antiqua (89). Microtus and pestoides belong to Y. pestis, but are atypical Y. pestis strains. They cause disease in rodents, but are avirulent to larger mammals like humans. Unlike the three classical biovars they can ferment rhamnose and melibiose (pestoides) or cannot reduce nitrate or ferment arabinose (microtus). (16, 400) All human pathogenic Yersinia species share a common virulence plasmid that encodes for the so called type III secretion system (T3SS). As Yersinia pestis has evolved, it has adopted yet another pair of plasmids, termed pPla/pPCP1 (9.6 kb) and pFra/pMT1 (102 kb). These two additional plasmids encode for proteins that function in tissue invasion, infection of the flea vector, and capsule formation. (56, 150) The

murine toxin (Ymt) of pFra has phospholipase D activity and improves survival in the flea midgut. Moreover, plasmid pFra encodes for the fraction 1 (F1) capsule-like antigen thought to be of importance for the antiphagocytic capacity of Y. pestis. Plasmid pPla encodes for a plasminogen activator (Pla). (62, 132, 150) Intriguingly, the two additional plasmids cannot explain the increased virulence of Y. pestis (56, 86). In addition, a selection of genes in the Y. pestis genome has been inactivated and exists as pseudogenes. The gene inactivations might be of importance for the life cycle of Y. pestis. Examples of disrupted genes are the putative insect toxins (tcaA and tcaB). With the flea Xenopsylla cheopis as vector, the need for these genes is superfluous. In addition, YadA and Inv (invasin) are inactivated in Y. pestis. These two proteins are important for Y. enterocolitica and Y.pseudotuberculosis in adherence and invasion of mammalian cells. Y. pestis has no need for such adherent and intrusive proteins due to its evolved lifestyle that differs from the other Yersinia enteric species (see 1.2.2). (291, 319, 387) Furthermore, the lipopolysaccharide (LPS) has undergone changes with evolution. Yersinia pestis has rough LPS and Yersinia pseudotubersulosis has smooth LPS. The differences are the result of silenced O-antigen gene cluster in Yersinia pestis, a selection that possibly has occurred through the vector-dependent life cycle of Y. pestis. (325)

1.2.2 Diseases and symptoms coupled to Yersinia - Plague and Gastroenteritis

Plague outbreaks caused by Y. pestis can be grouped into three pandemics; Justinian’s plague (541-767 AD), the Black Death (1346 to the 19th century), and the third pandemic in China in the mid-19th century (4). The three Y. pestis biovars are specifically coupled to each pandemic. Biovar Orientalis corresponds to the third pandemic, Mediaevalis to Black Death, and Antiqua to Justinian’s plague. Occasionally, Y. pestis is transmitted to humans by the bite of an infected Xenopsylla cheopis flea that has fed on a rodent reservoir, or by aerosols. Y. pestis infections can cause bubonic and pneumonic plague with high death rates. (265, 387) Plague is often considered an overcome problem, but the third pandemic is still ongoing. During 2004-2009, 12503 cases of plague were reported and 843 people died of the infection. The infections were reported in 16 contries in Asia, Africa and the Americas. (371) Multidrug resistant (MDR) Y. pestis has emerged and has been isolated from patients in Madagascar (118, 119, 141). This shows the importance of finding new antibacterial targets that can act to prevent fatal Y. pestis infections. Y. pseudotuberculosis and Y. enterocolitica cause a much milder disease than Y. pestis. Unlike Y. pestis, both pathogens are ingested via contaminated water or food. In the small intestine, they cross the M-cells of

the Peyer’s patches, reach the mesenteric lymph node (MLN) and can disseminate to liver and spleen. Infections with the enteric Yersinia very rarely go systemic in healthy individuals (387). The infection symptoms (abdominal pain, diarrhea, fever, and vomiting) are associated with gastroenteritis and mesenteric lymphadenitis (278, 326).

1.2.3 Yersinia species and their intracellular nature

Regardless the differences in entry to the human host all three human Yersinia species have a tropism for lymphatic tissues, a hallmark of Yersinia infections (21, 40, 326). All three species have the ability to replicate within macrophages in vitro as well as in vivo (55, 105, 117, 276, 356, 357). The ability of Yersinia to replicate inside macrophages is an observation often up for discussion. For Y. enterocolitica and Y. pseudotuberculosis this capacity seems restricted to certain serotypes and not to others (276, 277). The T3SS is involved in the human infection (71, 72). It is plausible to envision onset of a Yersinia infection with an inactive T3SS virulence system that is turned on at a later stage of infection and that bacteria in an initial stage of infection are unable to prevent phagocytosis due to an inactivated T3SS. As earlier mentioned, Y. pestis enters the host via the flea vector or aerosols. The T3SS may for this reason not be active at the early stage of infection and it is reasonable to think that survival in macrophages is needed from the very start of infection. The enteric Yersinia species are ingested orally and an elapse of time in a T3SS activating milieu (37°C) activates the bacterial antiphagocytic response. (277) Even though these bacteria encounter the phagocytic cells with defense turned on, it has been shown that 50% of T3SS activated Yersinia still are phagocytosed by macrophages (287). It is important to bear in mind that the plasmid encoded T3SS is not required for intracellular replication, but for virulence (276). Nonetheless, the PhoP/PhoQ two-component system is required for replication in macrophages and a strain that lacks phoP is attenuated in virulence in the mouse model (135).

2. Secretion systems in gram-negative bacteria

In gram-negative bacteria a series of secretion systems that vary in complexity have been identified. The systems can be divided into systems that have evolved for secretion over the cytoplasmic (inner) membrane and those that have evolved for secretion over the outer membrane. The secretion systems allow the bacteria to secrete proteins over the membrane compartments, into the environment and into the host cell. The following sections (2.1-2.2) cover the basics of inner and outer membrane secretion and mechanisms for protein insertion in the inner membrane.

2.1 Secretion across the bacterial cytoplasmic membrane

Three pathways are utilized for secretion over the bacterial inner membrane: the Sec system (general secretory system), the SRP system (signal recognition particle) and the Tat (Twin-arginine translocation) system. Components for the Sec and SRP system can be found in all genomes so far sequenced (52). The type II secretion system (section 2.2.2) and the type V secretion system (section 2.2.4) only transport proteins over the outer membrane and are dependent on the Sec or Tat pathways for export from the cytoplasm to the periplasm.

2.1.1 The general secretory system (Sec)

The Sec system is found in bacteria, eukarya and archaea. Due to its essential role in membrane biogenesis, the Sec system is the only secretion system identified as required for life. (77, 270) The system recognizes a cleavable signal peptide in the N-terminus of preproteins predestined for Sec secretion over the inner membrane to the periplasmatic space. The signal peptide that distinguishes Sec targets from other cytoplasmic proteins is a 20-30 residue extension with at least one positive charge at the N-terminus and a hydrophobic core of 8-12 residues. The protein SecB binds to preproteins and targets them to the cytoplasmic membrane and the Sec complex composed by SecA and the membrane-bound Sec translocase (SecY, SecE and SecG). Here, SecB is released from the preprotein and SecA translocates the protein through the pore by ATP hydrolysis. Now, the signal peptide is proteolytically removed by a bacterial signal peptidase and a mature protein is generated. (99, 258, 388)

2.1.2 The signal recognition particle system (SRP)

Membrane insertion of most inner membrane proteins that have somewhat hydrophobic, uncleavable signal peptides occurs via the Sec-translocon (82). Unlike preproteins targeted for the Sec system, inner membrane proteins are targeted to the cytoplasmic membrane in a co-translational manner. This does not occur by use of SecB but by the signal recognition particle (SRP) and the receptor FtsY. The SRP consists of an RNA molecule and the GTPase P48 (also called Ffh, fifty-four homolog). The SRP binds to ribosome- nascent membrane proteins by binding to hydrophobic transmembrane segments in them. The SRP bound to the target interacts with FtsY. FtsY binds to membranes and also interacts with the SecYEG complex. FtsY interacts with Ffh and the nucleotide-binding affinity of both proteins changes which allows them to bind GTP. Upon GTP hydrolysis, the target partially synthesized polypeptide chain and the attached ribosome are

transferred to SecYEG and inserted in the cytoplasmic membrane. (93, 99, 146, 258) Worth mentioning is that not all inner membrane proteins are inserted in the membrane via the SRP/Sec system. A few small integral membrane proteins are targeted to a protein called YidC and are inserted in the cytoplasmic membrane in a Sec-independent manner. Still, YidC is dependent on the SRP, SecA and SecYEG for its own insertion in the IM (191).

2.1.3 Twin-arginine translocation (Tat)

The Tat system is found in cytoplasmic membranes of bacteria and archaea and in thylakoid membranes of plant plastids (244). Unlike the Sec system, Tat translocation is not essential for life. In E. coli the translocon is encoded by the tatABCD operon and TatE (283). TatD is a cytoplasmic DNase redundant for Tat translocation (370). TatE encodes for a TatA paralogue. Studies in E. coli have put forth that the Tat system mainly consists of a TatABC complex that interacts with fully folded proteins that have an N- terminal twin-arginine leader motif, RRXΦΦ. The arginines are not replaceable and the Φ indicates hydrophobic residues. The energy for protein translocation by the Tat translocon comes from the proton motive force (PMF). (75, 283) In Yersinia the Tat translocon is encoded by the tatABCD operon (200). Yersinia depleted of TatC is attenuated in the systemic mouse infection model which shows that Tat secretion is important for Yersinia virulence (200).

2.2 Secretion across the outer membrane

There are seven described secretion systems that act to deliver substrates over the outer membranes of bacteria. Type III secretion in general and specifically T3S in Yersinia will be described in detail later (2.3-2.7.2). The type VII secretion (T7SS) is unique to Mycobacterium. Mycobacterium is not in true sense a gram negative so T7SS will not be dealt with in this section. (1, 341) Type I, type III, type IV and type VI secretion delivers proteins over both bacterial membranes in a periplasmic intermediate independent manner. Secretion systems III, IV and VI are capable of direct transfer of effector proteins over the target cell membrane into the target cell cytosol via a process called translocation. The following passages give an insight into Sec- and Tat-dependent and independent secretion over the outer membrane. Figure 2 illustrates the general specifics of the secretion systems described in 2.2.1-2.2.6 and in section 2.3-2.7.2.

2.2.1 Type I secretion (T1S)

The type I secretion system (T1SS) is built up by three main components: an inner membrane ATP-binding cassette (ABC) protein, an outer membrane protein (OMP) with pore-forming capacity and a membrane fusion protein (MFP). The MFP acts to link the systems outer and inner membrane components. T1S allows for a one-step secretion without periplasmic intermediates. Secretion through the system is Sec-independent and the ATP hydrolysis capacity of the ABC transporter energizes the translocation. Substrates are recognized by loosely conserved secondary structures in their C-terminus. Some examples of virulence determinants secreted by the T1SS are: The toxin CyaA of Bordetella pertussis (The causative agent of whooping cough), α-hemolysin (HlyA) of some uropathogenic Escherichia coli (UPEC) strains and the RtxA toxin from Vibrio cholera. (85, 127)

2.2.2 Type II secretion (T2S)

The type II secretion system (T2SS) is situated in the inner and outer membranes of the bacterium and is dependent on the Sec or the Tat systems. Secretion of proteins by the T2SS occurs in two consecutive steps. First proteins are translocated over the inner membrane by the Sec or Tat pathways. Then, the proteins are exported from the periplasm by a secretin situated in the OM. The T2S machinery is believed to have evolutionary ancestry from the type IV pilus. The T2SS consists of 12-15 core components that in short can be grouped as: the outer membrane secretin (protein D), a cytoplasmic ATPase and an inner membrane protein complex that extends to the periplasmatic compartment. The N-terminus of the secretin is thought to recognize substrates. Some secretin proteins are associated with secretin- specific lipoproteins (lipoprotein S) that guide and insert the secretins into the outer membrane. (65, 173, 194, 355, 386) Yersinia enterocolitica carries two T2SSs; yts1 and yts2. System yts1 is important in mouse virulence (169). Two examples of substrates secreted by T2S are the cholera toxin (Vibrio cholerae) and exotoxin A (Pseudomonas aeruginosa) (65).

2.2.3 Type IV secretion (T4S)

The multi-component type IV secretion system (T4SS) is related to the bacterial conjugation machinery. T4SSs are grouped according to their distinct functions (DNA uptake, conjugation and effector translocation). The unique ability of the T4SS to transfer both proteins and DNA distinguishes it from all other secretion systems. By conjugation, T4S mediates horizontal gene transfer. The structure of the system spans both bacterial membranes and is in general finished off with a pilus-like structure. The structure can

interact with eukaryotic cells and deliver DNA or proteins directly into the cell. Often as many as three ATPases power the secretion system. The plant pathogen Agrobacterium tumefaciens delivers proteins and the Ti plasmid into the host with its T4SS. (116, 355)

2.2.4 Type V secretion (T5S)

T5S is divided into three groups: autotransporters (AT or type Va), two- partner secretion (TPS or type Vb) system, oligomeric coiled-coil adhesion (Oca or type Vc) (87). The first step in T5S is Sec- or Tat-dependent. Autotransporters are characterized by their simple OM insertion. ATs have three domains: an N-terminal secretion signal recognized by the Sec- system, a passenger domain (α domain), and a C-terminal β domain. After IM passage, the C-terminus inserts in the OM and enables passenger domain export to the bacterial exterior. The passenger domain may be released from the membrane or stay attached to it. The first identified autotransporters are the IgA proteases of Neisseria and Haemophilus (269, 274). The TPS system consists of secreted proteins termed TpsA and the OM transporter, β-barrel, accessory protein TpsB. TPS is typically coupled to secretion of large virulence proteins. In a Sec-dependent fashion, TspB and TspA cross the IM. Interaction between TspA and the cognate TspB protein is cruicial for export over the OM. After OM transport, TspA may stay attached to the OM or be realesed to the external environment. TspA proteins fold at the cell surface. (194, 348) TpsA protein functions range from hemolysis (SlhA of Serratia marcescens), adhesion to host cells (HrpA of Neisseria meningitidis) and iron acquisition (HxuA of Haemophilus influenzae) (172, 308). An example of oligomeric coiled-coil adhesion is the nonfimbrial YadA adhesin of Yersinia. Oca proteins are surface attached oligomeric ATs that form oligomeric complexes at the bacterial surface. The features of Oca proteins are: an N-terminal Sec-dependent secretion signal, a conserved neck domain, a stalk domain, and a C-terminal membrane anchor domain with a coiled-coil segment. (285) Proteins of this group bind to eukaryotic cell surfaces (NadA of Neisseria meningitidis) and to extracellular matrix (ECM) proteins (HadA of Haemophilus influenza) (53, 313).

2.2.5 The chaperone/usher (CU) pathway

Various adhesive surface structures are assembled by the Sec-dependent CU pathway. The secretion system consists of a periplasmic chaperone and an integral OM usher protein. Substrates of the CU system fold in a chaperone assisted manner in the periplasm before they cross the OM. (194) Surface structures assembled by the CU pathway are: type 1 and P pili from

europathogenic E. coli and the capsule-like F1 and pH6 antigens of Yersinia pestis (94, 347, 348).

2.2.6 Type VI secretion (T6S)

Type VI secretion systems (T6SS) consist of protein complexes that span both bacterial membranes. T6S is found in a wide variety of bacteria including human pathogens, plant and soil bacteria. T6S has a role in biofilm formation, stress sensing and pathogenesis. Hcp (hemolysin coregulated protein), VgrG (valine-glycine repeat protein) are proteins secreted by the system. (27) EvpC (Hcp-like), EvpI (VgrG), and EvpP are secreted by the Edwardsiella tarda T6SS (399). No Sec- or Tat- secretion signal has been identified for T6 substrates. The T6 machinery components are a putative lipoprotein, IcmF, IcmH (DotU), and the energizer ClpV AAA+ ATPase. (27, 54) It is not uncommon that bacteria carry genes for more than one T6SS in the genome (28). For instance Yersinia pestis has five T6SS loci (104).

Figure 2. Graphic illustration of secretion systems I-VI, denoted T1-T6SS in the figure. T1S-T4S and T6S span both bacterial membranes. T5S occurs specifically over the outer membrane (OM). T2SS and the T5SS rely on the Sec or the Tat secretion system for the transport of substrates over the bacterial inner membrane (IM). T3SS, T4SS and T5SS are able to transfer substrates in a one step process over the host cell membrane (CM). The T4SS can transport both proteins and DNA. P indicates the periplasmic space. For more details see 2.2.1-2.2.6 and 2.3-2.7.2.

2.3 The Type III secretion system (T3SS)

The T3SS is a multi-protein structure that spans both membranes of gram- negative bacteria. Secretion by the system is Sec-independent, thus it does not generate cleaved secretion signals or periplasmic intermediates. On the other hand, some of the components that build up the secretion apparatus rely on the Sec-system (47, 160, 318). Due to the predominant location of

T3SS genes on pathogenicity islands (PAIs) and on plasmids it is believed that the clustered genes are acquired by horizontal gene transfer (70, 134).

Table 1. The seven different T3SS families. Adopted from (70).

Family Species Description Hrp1 Pseudomonas syringae Plant pathogen Erwinia amylovora Plant pathogen Pantoea agglomerans Environmental and human commensal Vibrio parahaemolyticus Human pathogen Hrp2 Burkholderia pseudomallei Human pathogen Ralstonia solanacearum Plant pathogen Xanthomonas campestris Plant pathogen Rhizobium Rhizobium Plant symbiont Mesorhizobium loti Plant symbiont SPI-1 Salmonella enterica Human pathogen Shigella flexneri Human pathogen Burkholderia pseudomallei Human pathogen Chromobacterium violaceum Human pathogen Yersinia enterocolitica Human pathogen Sodalis glossinidius Symbiont (Tse-Tse fly) SPI-2 Escherichia coli EPEC Human pathogen Escherichia coli EHEC Human pathogen Salmonella enterica Human pathogen Citrobacter rodentium Mouse pathogen Chromobacterium violaceum Human pathogen Yersinia pestis Human and rodent pathogen Yersinia pseudotuberculosis Human and rodent pathogen Edwardsiella tarda Human pathogen Ysc Yersinia pestis Human and rodent pathogen Yersinia pseudotuberculosis Human and rodent pathogen Yersinia enterocolitica Human pathogen Vibrio parahaemolyticus Human pathogen Bordetella pertussis Human pathogen Pseudomonas aeruginosa Animal, insect and human pathogen Desulfovibrio vulgaris Environmental bacteria Photorhabdus luminescens Mutualistic bacteria Aeromonas salmonicida Fish pathogen Chlamydiales Chlamydia trachomatis Human pathogen Chlamydia pneumoniae Human pathogen

The T3SS apparatus is equipped with a cytoplasmic ATPase and an extracellularly located structure often referred to as the needle. By use of the T3SS bacteria secrete proteins to the extracellular environment as well as inject proteins into the target cells, a process called translocation. The functions of the translocated proteins are often to mimic common functions in the target cell and disrupt cellular functions.T3SSs are found in a wide variety of bacteria ranging from human pathogens to plant pathogens as well as symbionts and are grouped into seven families (Table 1) (160, 305, 379). Examples of diseases connected to T3SSs are: plague, whooping cough, pneumonia and enterocolitis. Some bacteria encode for more than one T3SS. For instance, Salmonella has two pathogenisity islands each encoding for one secretion system. Salmonella pathogenicity island 1 (SPI-1) is important for the initial stages of the infection (penetration of the intestinal mucosa) and Salmonella pathogenicity island 2 (SPI-2) is required for the later systemic stages of the infection. Although the flagellum and the T3SS share common traits, they phylogenetically group into different families (110).

2.4 The flagellar T3SS

The bacterial flagellum and the T3SS are evolutionary closely related. The similarities between the flagellar- and the injectisome-T3SS are seen on level of function, structural resemblance and sequence (7, 33, 160, 216). Still, the scientific community has not been able to establish if the flagellum derived from the T3SS or vice versa, or if both secretion systems have a common ancestor (300). Nonetheless, the relatedness is apparent and extends to acceptance of substrates destined for other secretion systems than their own (204, 393, 394). The flagellum of Salmonella typhimurium has been extensively studied and is involved in motility. The multi-protein construction is built up by three main structures: (i) a basal body inserted in the inner and the outer membrane and a rod that transverses the periplasmatic compartment. (ii) A hook that connects the basal body to (iii) the filament (25, 217). The flagellum assembly is strictly regulated on transcriptional level, assembly level and also has a built in switching mechanism (2.4.1) (64). The assembly starts with the formation of cytoplasmic base of the basal body (the C- and MS-rings). The basal body encompasses a flagellar-specific T3SS (8, 231). The rod is the next structure constructed, followed by the L- and P-rings in the LPS and peptidoglycan and compartments. Next follows the hook assembly which connects the basal body to the flagellum filament. The external hook, rod and filament structures are exported to the bacterial surface by use of the T3SS assembled in the basal structure. (63, 96, 165, 180, 230, 231) An example of transcriptional regulation is the regulation of late exported flagellin by the anti-σ28 factor FlgM. (128, 129) Once the hook-basal body (HBB) structure

has been finalized, FlgM is release from σ28 and secreted from the bacterium and transcription and assembly of flagellin is initiated and the flagellar structure is finished (161, 197). The actual regulation of flagellin (FliC) secretion occurs via a sensory timing mechanism called the substrate specificity switch (377). The substrate switch regulation is conducted by two proteins: FliK and FlhB. The switch occurs as the hook is fully assembled and initiates FliC secretion. Before flagellin export, the wild-type hook develops a specific length of 55 nm (151). Studies have pointed out a series of regulatory models for hook length regulation and substrate specificity switching: the internal ruler model (316), the molecular ruler model (183), the molecular clock model (236), the measuring cup model (219). For simplicity, the following section will entail overall details on FliK and FlhB co-regulation only.

2.4.1 FliK and FlhB – The flagellar substrate specificity switch

FlhB is a 42 kDa cytoplasmic protein with an integral transmembrane portion which anchors the protein to the cytoplasmic membrane (229). A feature important for the regulatory function of FlhB is displayed in the C- terminal cytoplasmically located tail. Here, the so called NPTH (Asparagine- Proline-Threonine-Histidine) motif is found. This motif is conserved in all T3SS and flagellar orthologues (201, 232, 397). The protein has been reported to be exposed to an autocleavage mechanism in the conserved site. Specifically, cleavage of FlhB occurs at Proline-270 and generates the cleaved products FlhBCN and FlhBCC (232). FlhBCC formation can be monitored as a cleaved product of about 11 kDa on an SDS-PAGE. Cleavage occurs at a higher efficiency at higher pH and FlhBCN and FlhBCC interact with each other. (102, 232) Moreover, processing of FlhB occurs in a self-cleavage event independent of a responsible cleavage protease (102). FlhB has been coupled to needle and hook length regulation through studies of yet another regulatory protein: FliK. FliK has a key regulatory function in hook-length control. The wild-type hook is about 55 nm long whereas a fliK deletion mutant displays poly-hook structure with lengths that can reach up to 900 nm (151, 262, 338). A fliK mutant has no filament attached to the abnormally lengthy hook, hence it is unable to perform the substrate specificity switch (151). FliK acts to sense the finished hook and stops hook production in advantage for filament assembly (198, 228, 241, 377). The regulatory coupling between FliK and FlhB comes from the discovery that mutations in FlhBCC suppress the ΔfliK poly-hook phenotype (198, 377). The second site suppressor mutations introduced in FlhB in the fliK deletion mutant still show poly-hooks, but do assemble filaments and are able to use the flagellum for movement. It is likely that FlhB regulates the switching mechanism with FliK by an induced

conformational change rather than by the FlhB cleavage per se (112). In addition, FliK interacts with both FlhBC and FlhBCC (232).

2.5 The Yersinia T3SS

Unlike the flagellum, the Yersinia T3SS is not a structure involved in motility. This T3SS is tailored for specific targeting and translocation of effector proteins over the bacterial membranes and the eukaryotic cell membrane into the host cell cytosol. The T3SS of Yersinia is encoded on a virulence plasmid. The plasmid carries all genes required for the secretion apparatus, the secreted proteins, proteins needed for translocation into the host, and T3SS regulatory proteins. The system is absolutely required for virulence and T3SS mutants are attenuated in mouse infection models. Additionally, Yersinia also encodes for a chromosomal T3SS. Unlike the plasmid encoded T3SS, the chromosomal T3SS has only been connected to virulence in Yersinia enterocolitica (142, 276). The following sections scrutinize the plasmid encoded T3SS of Yersinia and figure 3 shows a very simplified model of Yersinia T3SS build up, protein export and regulation.

2.5.1 The type III secretion apparatus – Ysc proteins

The T3SS is often referred to as the syringe, the injectisome or the nanomachine. It is inserted in the IM and the OM, connected by a rod and topped by an external structure protruding from the bacterium. The T3SS is capable of protein secretion into the extracellular milieu and protein translocation over the bacterial membranes and the target cell membrane into the target cell cytosol. (Figure 3A) A collection of about 20-some ysc (Yersinia secretion) genes are responsible for the making of the T3SS in Yersinia. The genes are gathered in operons denoted virA, virB, virC and virG where vir stands for virulence (Table 2). The transcriptional regulation of the operons is discussed later (see 2.6.1). The genes of the T3SS are activated by signals like lowered calcium concentrations (in vitro), cell contact (in vivo), and elevated temperature (37°C). Three of the structural proteins are secreted via the Sec-system: (i) the secretin YscC, (ii) the secretin pilot lipoprotein YscW and (iii) the potential membrane-linker YscJ (47, 225, 318). The other components find their correct localization in a Sec- independent manner. It is possible that secretion of external structures occurs through secretion by the T3SS itself. A structure called the needle forms a 60-80 nm hollow extracellular structure about 7 nm wide with an internal tube of approximately 2 nm width (155). The energy to drive secretion by the T3SS most likely comes from the ATPase YscN, the energizer (31, 381). The Ysc proteins are more than building blocks of the secretion machinery. For example two Yscs have been assigned regulatory roles in the

secretion process as such and in sensing of completion of the T3SS apparatus assembly (YscU and YscP) (12, 95, 359, 360). A final remark is that actual secretion through the needle structure is hypothesized, but has never been observed. Recently, it was shown in Yersinia that effector protein translocation can occur by a surface-localized translocation-competent intermediate. This argues for a two-step transfer of effectors, rather than a one-step translocation event. (10) The needle and the secretin have been successfully visualized by electron microscopy in Yersinia, but not the complete secretion apparatus (155, 195). The structures of type III secretion apparatuses of Salmonella typhimurium, Enteropathogenic Escherichia coli (EPEC) and Shigella flexneri have been successfully visualized by transmission electron microscopy (TEM) (196, 312, 342).

Table 2. Ysc gene operons virA and B and the encoded proteins with sizes, characteristics, localizations and information regarding whether the protein is needed for secretion (Y) or not (N). OM=outer membrane. IM=inner membrane. CP=cytoplasm. (12, 26, 30, 31, 42, 47, 81, 90, 95, 103, 166, 171, 195, 263, 268, 282, 310, 318, 330, 336, 385)

Required for Size Protein Characteristic Localization Yop (kDa) secretion Operon virA

YscX 13.6 Interacts with YscY. Secreted Y YscY 13.1 Possible YscX chaperone. CP Y Interacts with LcrH. YscV/LcrD 77.8 Structural component. Protrudes IM Y into CP. Operon virB

YscN 47.8 ATPase IM/CP Y YscO 19.0 Interacts with YscP. Secreted Y YscP 50.4 Interacts with YscQ, YscN, YscL Secreted Y and YscO. Involved in the substrate specificity switch. Regulates the needle. YscQ 34.4 Interacts with YscL and YscK. CP Y YscR 24.4 Structural component. Middle IM Y part of protein in CP. YscS 9.6 Structural component. IM Y YscT 28.4 Structural component. IM Structural component. Involved in YscU 40.4 the substrate specificity switch. IM Y Protrudes into CP.

2.5.2 Type III effector proteins

Despite the intracellular survival capacity of Yersinia, its T3SS effectors are tailored to avoid phagocytosis by macrophages and polymorphonuclear leukocytes (PMNs) and act to subvert the action of the host immune response (32, 295).

Table 2, continued. Ysc gene operons virC and G and the encoded proteins with sizes, characteristics, localizations and information regarding whether the protein is needed for secretion (Y) or not (N). OM=outer membrane. IM=inner membrane. CP=cytoplasm.

Required Size Protein Characteristic Localization for Yop (kDa) secretion Operon virG

YscW 14.6 Lipoprotein. Pilots YscC to the OM. OM Y Stabilizes the secretin. Secreted via Sec. Operon virC YscA 3.8 Unknown Unknown N YscB 15.4 Forms a heterodimeric chaperone CP Derepressed for YopN with SycN. secretion YscC 67.1 Interacts with YscD. Secreted via OM Y Sec. Forms the oligomeric secretin ring. YscD 46.7 Connects the membrane rings. IM/PP Y Forms MS-ring. Interacts with YscC and YscJ. YscE 7.4 Interacts with YscG. YscF CP/IM Y chaperone. YscF 9.4 Needle component. Surface Y YscG 12.9 YscF chaperone. CP/IM Y YscH/YopR 18.3 Controls YscF secretion or YscF Secreted N polymerization YscI 12.6 Regulated by YscU and YscP. Secreted/CP Y YscJ 27.0 Lipoprotein. Secreted via Sec. CP/IM/PP Y Possible membrane-linker. Forms MS ring. Interacts with YscD. YscK 23.9 Interacts with YscQ. CP Y YscL 24.9 Interacts with YscN and YscQ. CP Y

Yersinia has six effector proteins called Yops (Yersinia outer proteins). The effects and activities of all Yop effectors are summarized in table 3. All together the proteins act to promote the extracellular lifestyle of Yersinia. YopE, YopH, YopT and YpkA (YopO in Yersinia enterocolitica) are involved in the antiphagocytic response. YopM and YopJ (YopP in Yersinia enterocolitica) are involved in immune response modulation. (177, 353, 372) YopT is dispensible for virulence, whereas YopE, YpkA, YopM and YopH are essential for virulence. The role for YopJ in virulence is unclear. (49, 120, 121, 206, 233, 353, 354, 372) As YopE is translocated into HeLa cells, it mediates a so called cytotoxic response. This is due to the GAP activity of YopE that depolymerizes actin and this effect is easily monitored as cell rounding (288-290, 384). YopK (YopQ in Yersinia enterocolitica) is unique to the Yersinia species. It is secreted and translocated by the T3SS, but it has no clear assigned effector function. Still, YopK is required for Yersinia to cause a systemic infection in both orally and intraperitoneally infected mice (158). YopK works as a regulator of translocation. A mutant that lacks yopK overtranslocates Yop effectors and overexpression of YopK reduces Yop translocation. Moreover, YopK regulates the pores formed by YopB and YopD. Consistent with the overtranslocating phenotype, a yopK mutant creates larger pores in infected erythrocytes. (39, 157)

Table 3. The effectors of Yersinia and their mode of action and activities. (15, 98, 120, 138, 140, 167, 176, 234, 255, 257, 288, 303, 314, 372, 375, 384)

Effector Mode of action Activity

YopE Anti-phagocytosis GAP YopH Anti-inflammation. Anti-phagocytosis PTPase YopJ Induction of anti-inflammation. Apoptosis Cysteine Protease of macrophages YopM Eukaryitic cell cycle interference Leucin-rich Repeat Protein YopT Anti-phagocytosis Cysteine Protease YpkA Anti-phagocytosis Serine-Threonine Kinase

2.5.3 Translocator proteins of Yersinia

In Yersinia, there are three proteins denoted as translocator proteins, namely YopD, YopB and LcrV. The translocator proteins are secreted by the T3SS and are involved in the transport (translocation) of effector proteins from the T3SS over the eukaryotic cell membrane into the host cell cytosol. Hydrophobic domains in both YopB and YopD point to membrane spanning capacities of both proteins (163).YopD and YopB, along with YopK, have roles in pore formation in infected erythrocytes (114, 157, 164, 250). YopB

and YopD also have the ability to form pores in macrophages and to insert into artificial bilayers (250, 343). Pores are yet to be identified in vivo in cells targeted during infection. LcrV was the first identified virulence protein of Yersinia. It was identified in the mid-1950s and is known for its protective immunity against plague. (17, 48) LcrV connects to the pore in such a way that it determines the size of the pore formed (156). In addition to pore regulation, LcrV is involved in the low calcium response (see 2.6.2). An lcrV mutant is unable to secrete YopB and YopD, but secretes other Yops. This finding, and the fact that LcrV interacts with YopD and YopB, points to a role for LcrV in YopD and YopB release from the bacterium. (301) In its secreted form, LcrV associates with the tip of the needle and suppresses the host immune response (239, 246, 247, 249, 369). Specifically, LcrV suppresses the immune response by signaling via the macrophage toll-like receptor 2 which leads to interleukin 10 induction and lowered TNF-α and IFN-γ production (41, 322). In the bacterium, LcrV has a role in inhibition of negative regulators such as YopN/LcrE, LcrG and LcrH (324). YopB is not required for secretion, but (as described above) has a role in translocation and pore formation (164). YopB also triggers a proinflammatory response in the host cell. This effect of YopB is puzzling, since this response is later counteracted by the other translocated Yersinia effectors (YopE, YopH and YopJ). (373) On top of the earlier described regulatory functions of YopD, YopD has additional regulatory roles. YopD is translocated into HeLa epithelial cells where it may interact with translocated YopE (111, 144). Furthermore, YopD (in cooperation with LcrH and LcrQ) acts to posttranscriptionally negatively regulate Yop expression by binding to the 5’ untranslated region of yop mRNA (13, 51).

Table 4. Effector and translocator proteins and their assigned chaperones. (108, 167, 205, 251, 366)

Protein Chaperone YopE SycE/YerA YopH SycH YopJ None YopM None YopT SycT YpkA SycO Translocators YopB and YopD LcrH

Figure 3. Schematic drawing of the Yersinia T3SS and proteins secreted/translocated by it. A. The T3SS spans both bacterial membranes and an external structure called the needle (made up by YscF monomers) protrudes from the bacterial surface. Secretion occurs to the surrounding, whereas translocation transfers proteins over the host cell membrane. The stars indicate Yops and LcrV that are secreted by the T3SS. B. Western blot analysis of culture supernatants from wild-type Yersinia. Depletion of calcium from the culture media results in an upregulation of Yop/LcrV expression and secretion. Total Yop and LcrV antibodies were used.

2.5.4 Chaperones of the Yersinia type III secretion system

For most of the effector proteins, chaperones are needed for proper function. The chaperones can be grouped into three classes: (i) the ones that chaperone the effector proteins, (ii) chaperones destined for the pore forming protein, (iii) chaperones that assist in secretion of proteins that polymerize outside the bacterium. Chaperones of Class I can be further divided into Class IA and Class IB, where Class IA binds one effector and Class IB binds multiple effectors. (261) Chaperones of the flagellar system are typically grouped in Class III (24, 256). The roles of the chaperones are to confer stability, maintain effectors in a secretion competent unfolded state, to prevent premature aggregation or to prevent degradation (58, 61, 74, 100, 331). Of the effectors YopE, YopH, YopT and YpkA have dedicated chaperones whereas YopM and YopJ do not (Table 4). Of the translocators, YopB and YopD are chaperoned, both by the chaperone LcrH (251). Two secreted negative regulators are also chaperoned: LcrQ by SycH, and YopN by YscB and SycN (Syc, specific Yop chaperone) (80, 170, 389). One last proposed role of chaperones is the ability to work as secretion signals guiding the protein to be secreted to the T3SS (29). The ATPase YscN has the ability to bind the secretion signal (2.5.5) of the secreted protein YopR (328). An YscN homolog (InvC) in the Salmonella typhimurium T3SS has the capacity to dissociate chaperones from its cognate effector and bind the effector (9). This event is seen in EPEC too, where the ATPase EscN interacts

with both the chaperone CesT and its assigned effector Tir (123). This illustrates a role for T3SS ATPases in substrate recognition, effector chaperone dissociation and maintenance of effectors in an unfolded state. Likely this occurs in a common recognition mechanism. A final remark is the fascinating finding that chaperones can be recognized by heterologous T3SSs. This was demonstrated in Xanthomonas campestris that has the ability to secrete Yersinia YopE in a YerA-dependent manner by its own T3SS (293).

2.5.5 Type III effector secretion signals

Not only do the effectors rely on the T3SS for their export, a subset of the Ysc proteins also do (2.5.1 and table 2). Although no conserved motif has been found that unites all T3SS secreted proteins, a short stretch of the N- terminus provides secretion competence. An amphipathic nature of the secretion signal seems advantageous and the signal was first suggested to lie in the mRNA and later shown to be in the protein sequence. (14, 211-213, 226, 329)

2.6 Regulation of the Yersinia T3SS

Regulation of the T3SS of Yersinia is a complex event that requires elements such as temperature, calcium/target cell contact and a transcriptional regulator. This chapter aims to give an overview to the field of Yersinia T3S regulation.

2.6.1 The role of temperature

LcrF (VirF in Y. enterocolitica) is a transcriptional activator and regulatory protein that belongs to the AraC regulatory protein family (69). Depending on Yersinia strain regulation of and by LcrF differs. In Yersinia enterocolitica the negative-regulatory histone-like protein YmoA binds to and represses the lcrF promoter at lower temperatures (73). Higher temperatures (37 °C) melts bends in the virulence plasmid, including in the lcrF promoter, and activates lcrF synthesis (286). LcrF in turn activates the T3SS genes. In Yersinia pestis, on the other hand, the thermoregulation on LcrF production appears more significant on a translational level (153). Here transcription of lcrF is temperature-independent (154). The Shine-Dalgarno sequence of transcribed lcrF is molded in a stem-loop structure. Increased temperature (37°C) destabilizes the stem-loop structure and LcrF is translated and T3SS gene transcription is induced. (153, 199) LcrF activates virC and yop transcription, but is not essential for induction of virA and virB activation (199). Operons virA and virB do respond to temperature and the

expression of genes from these operons is elevated at 37°C. The virB proteins are needed for full expression of yop genes and yadA (225). A final remark is that YmoA is identical in both Yersinia strains so the differences in thermoregulation are somewhat of a mystery (73). The mechanism of LcrF regulation in Yersinia pseudotuberculosis is still unsolved.

2.6.2 The low calcium response (LCR)

The low calcium response in Yersinia describes how in vitro lowered calcium levels induce the T3SS, growth restriction, and T3S substrate expression and secretion (106, 130). In vitro the Yersinia T3SS is repressed by mM concentrations of calcium (2.5 mM) and calcium depletion triggers T3SS expression and secretion (Figure 3B). Low calcium triggering of T3SS also occurs in for instance Pseudomonas aeruginosa (390). The Shigella flexneri T3SS is induced by Congo red (18). The mM calcium environment is also required for growth at 37°C, but not at 26°C. Growth restriction upon calcium depletion and temperature of 37°C occurs as a result of defects in RNA stability and protein synthesis. RNA instability and lowered protein synthesis does not include Yops. (57, 396) Wild-type Yersinia displays calcium dependent (CD) growth phenotype with Yop expression and secretion upon depletion of Ca2+ at 37°C followed by growth arrest (Figure 3B) (125, 149). Yersinia devoid of the virulence plasmid no longer relies on calcium for growth and display a calcium independent (CI) growth phenotype (273). A CI growth phenotype includes no growth restriction regardless calcium concentration or increased temperature to 37°C. Mutations in positive regulatory genes, and a good number of the ysc genes, show CI growth phenotypes. (69, 109, 392) The last group of strains that can be connected to calcium is the temperature sensitive (TS) or ‘calcium-blind’ mutants. At 37°C this group of strains is unable to grow regardless calcium concentration. (109, 240, 275, 392) TS strains are unable to exert negative regulation on Yop expression and often allow secretion in the presence of calcium (280). (72, 392) The precise function of calcium regulation on the Yersinia T3SS is not known, but calcium works as an efficient tool for laboratory understanding of T3SS regulation and function.

2.6.3 Other Yersinia T3SS regulatory elements

A collection of proteins have been directly linked to negative regulation of Yops. The protein LcrQ is connected to negative regulation of Yop transcription in connection with the chaperone LcrH and YopD. YopN, LcrG and TyeA, on the other hand, are directly linked to Yop release (59, 84, 107, 168, 222, 252, 302, 323).

YopN is expressed in both absence and presence of Ca2+ in the growth medium and is exposed on the bacterial surface in plus Ca2+ condition (107, 168). Upon calcium depletion, YopN is released from the bacterium. The external localization of YopN prior to Yop secretion onset has led to the idea of YopN as an outer gatekeeper of the T3SS. Regardless the mode of regulatory action, a mutant devoid of yopN secretes Yops to the surrounding environment despite calcium concentration in the growth medium or target cell contact. This leakage in Yops differs greatly from the polarized Yop translocation seen in the wild type, where Yops are not found in the surrounding cell environment upon target cell contact. (290) To add to the complexity; in the bacterium YopN associates with TyeA and with two chaperones, namely YscB and SycN (80, 168, 170). It is proposed that YopN, in association with its chaperones and specifically to TyeA, acts as a plug blocking Yop secretion from inside the cell. YopN release from TyeA opens the plug and allows YopN and other Yops secretion. (60, 79) TyeA is localized on the bacterial surface and in the cytosol and is associated to the translocation complex through interactions with both YopD and YopN (168). A tyeA mutant shows calcium blind, TS, phenotype and secretes Yops in the presence of calcium. The last protein LcrG is also required for proper Yop secretion and translocation (302). LcrG is mainly situated in the cytosol and associates with LcrV (252). In the cytosol LcrV may act to sequester LcrG away from the T3SS upon target cell contact. This would in turn further boost Yop expression. (222, 252) LcrG is at times called the inner gatekeeper due to its proposed regulatory role in T3S substrate export from the cytosol of the bacterium (252, 301). An lcrG mutant is calcium blind and TS, like the yopN mutant. LcrG interaction with either TyeA or YopN is unclear. Taken together; YopN, LcrG and TyeA act to block the secretion conduit from release of Yops prior to target cell contact or depletion of calcium from the growth medium.

2.6.4 Target cell contact dependent Yersinia T3SS regulation

For Yersinia target cell contact triggers T3SS substrate expression, secretion and translocation (267, 290). How the target cell dependent regulation takes place is not known. It is known that intimate contact between the target cell and the bacterium allows for secretion of the negative regulatory protein LcrQ. Release of LcrQ relieves the block of Yop expression. (50, 267, 280) As previously mentioned, the needle carries a tip-protein: LcrV (239). LcrV associates with the translocation pore, therefore it is likely that LcrV at the tip of the needle acts to sense target cell contact (156, 266). It is possible to view Tye, YopN or LcrG as sensing proteins. Mutations in either gene results in premature effector protein secretion (presence of Ca2+) (see section 2.6.3).

In this case lack of sensor would lead to a constitutively open needle complex. TyeA and YopN are extracellularly located and LcrG secretion has been reported (323). Although, secretion of LcrG is not of importance for its negative regulatory function on Yop release (279). One cannot rule out the possibility that one of the ysc gene products, which deletions result in secretion negative phenotypes, are involved in cell sensing. Lack of the sensor, in this scenario, leads to a constitutively closed needle complex. For instance, the needle has been implicated as the sensor that recognizes the target cell. In vitro, YscF is thought to mimic calcium levels resembling those of target cell contact. (34, 83, 238)

2.7 Secretion control performed by YscU and YscP

The assembly of needle structures, the secretion and translocation by the machines and the regulation of these events may differ depending on bacterial species. Indeed, there are several regulatory models that describe the control of effector secretion. One is the prediction of a sensor protein that senses target cell contact and opens up a premade plug the T3SS channel for release of effectors. A candidate sensor is YopN of Yersinia, MxiC of Shigella flexneri or PopN of Pseudomonas aeruginosa. (34, 36, 101, 107, 337) Another model describes how the structure on the needle tip adopts a closed and an open state blocking and allowing effector secretion, but more importantly translocation (34, 83, 237, 367). Other studies imply that the needle tip may confer conformational changes onto the T3SS apparatus which in turn allows for effector secretion (34, 185, 351). On another level YscU and YscP, along with 5 more genes of the virB operon, have been shown important for Yop secretion (See table 2). In greater detail YscU and YscP have been coupled to T3SS regulation in the same fashion as described for FlhB and FliK of the Salmonella flagellum (2.4.1). An yscP mutant is severely affected in the switch from secretion of the needle protein YscF to Yops and carries excess YscF on the bacterial surface as compared to the wild type (wt). Introduction of single site amino acid mutations in certain positions of YscU reverse the phenotype of the yscP mutant. These mutations in yscU introduced in the yscP mutant allow for Yop secretion to occur and lower the amounts of surface localized YscF. Hence, the switch problematic seen in the yscP mutant is overcome. (95) As for the FlhB/FliK proteins in the flagellum YscU/YscP of Yersinia are coupled to needle and Yop regulation in something termed the substrate specificity switch. It is described such that YscU and YscP coordinate sensing of needle completion and switch to Yop secretion initiation.

2.7.1 YscU and the conserved NPTH motif

YscU is the last protein of eight encoded on the virB operon (26). The protein is 354 amino acids long and can be divided into four distinct parts (Figure 4A). The first part encodes four transmembrane segments that anchor the protein to the IM of the bacterium (Figure 4B). Then follows a large cytoplasmic domain (YscUC) with a conserved site consisting of four amino acids: Asparagine-Proline-Threonine-Histidine (NPTH). (12) The NPTH motif confers cleavage ability and as a result the large C-terminus that lies in the cytoplasm can be further sub-divided into two domains: YscUCN and YscUCC (Figure 4A and B). Cleavage occurs specifically at position Proline-264 of the conserved motif and substitution mutations of N263 or

P264 to Alanine abrogate cleavage and no YscUCC is formed. Unprocessed and processed YscUC variants have successfully been crystallized and the structures point to differences in conformation depending on cleavage state (215, 374). Mutagenesis has also shown that YscU is essential for a functional T3SS, and so is the NPTH site (12, 201). All mutations in yscU that reverse the yscP phenotype are located in YscUCC (95). Furthermore, two studies have implicated a role for YscU processing in specific regulation of translocator secretion (281, 327).

Figure 4. Illustration of YscU. A. YscU can be divided in four parts. A transmembrane part (YscUTMS), a cytoplasmic part (YscUC). A conserved site (NPTH) is found in the cytoplasmic domain and here YscU is specifically cleaved at position Proline-264 which generates YscUCC and YscUCN. B. The four transmembrane domains anchor YscU in the inner membrane (IM) with YscUC protruding into the cytoplasm.

2.7.2 On the role of YscP

Different regulatory models have emerged and aim to describe how sensory mechanisms have developed to monitor maturation of the T3SS complex and allow for effector secretion. The inner rod model was formed by studies in Salmonella typhimurium and opposes the models described for YscP as a molecular ruler (see later in section) and sole sensor for T3SS completion. This model suggests that the maturation of the Salmonella needle structure is not what determines fitness, but rather the formation of the inner rod consisting of PrgJ (220). A separate study on the Yersinia inner rod protein YscI supports this finding (385). Yet another model that describes flagellar hook length control and initiation of the switch has been described (219). Here the C-ring of the flagellar basal body would pre-measure the hook subunits and rapidly export them. Then FliK would interact with FlhB and switch the export specificity to filament type. Mutational studies of the hook protein (FlgE) (YscF in Yersinia T3SS) of the flagellum put forth the need for correct timing of hook assembly and proper rate of hook elongation. In the study mutant FlgE proteins were examined that caused weak motility, delay in filament (FliC) assembly and shorter hooks. Overproduction of the mutant proteins, on the other hand, showed proper timing as seen with wt-like hooks. In the same study a C-terminal portion of FliK (FliKC) is reported as able to interact with the hook-cap protein (FlgD) and the hook protein per se. (236) On the whole, this places FliK in association with hook regulation and a timed process. YscP is a secreted protein needed for efficient effector translocation, but it is not itself translocated into eukaryotic cells (6, 264, 330). An yscP mutant shows a delay in cytotoxic response on HeLa cells (95). YscP is associated with the bacterial surface in the presence of calcium, whereas depletion of calcium releases YscP from the bacterium (6, 330). The export of YscP is crucial for needle length control (6). Two independent secretion signals have been reported for YscP (5, 6). Disruption of both signals is needed to block YscP secretion. Such a mutant YscP protein can still support Yop secretion, but displays deregulation of needle length. (6) The various existing YscP homologues in numerous T3SSs show little sequence conservation. Still, they all contain a conserved globular C-terminal domain essential for function. This domain can be interchanged in closely related YscP homologues (5). Export of YscP homologues, such as InvJ of Salmonella SPI-1 and FliK of the flagellum system, has also been documented as necessary for needle/hook length (228, 296). The most known function assigned to YscP is the ruler function. This function was discovered as shortened or elongated variants of YscP shorten or lengthen the needle (175). Further studies have connected YscP and the helical content of YscP to needle length and highlight the need of proper needle length for a functional T3SS (238, 359, 360).

The exact mechanism by which YscP performs its regulation on the T3SS remains a discussion, but one thing is certain: YscP is needed for a proper T3SS.

3. Bacterial stress response

All living organisms have different requirements for their existence. Environmental factors like osmolarity, temperature, pH and nutrient availability need to be fulfilled for outmost comfort and survival. For instance, some bacteria (For example Bacillus and Clostridium) have the capacity to enter a spore state upon nutritional starvation. The spore is a dormant state and can be reversed from its inactive state upon entry into a more nutrient favorable environment. As temperatures rise or drop other responses may be activated in the bacterium. For protection, heat shock systems are activated.

3.1 Heat shock response

Temperature, misfolded, and denatured proteins rapidly activate the cytoplasmic heat shock proteins (Hsps) and ATP-dependent proteases. Heat shock proteins are at times referred to as stress proteins. The response to heat stress protects the bacteria from dysfunctional misfolded proteins and protein aggregates. This is done by protein aggregate disaggregation, protein refolding and degradation. Two Hsp families have been implicated in the heat shock response; the Hsp60 (GroEL) and the Hsp70 (DnaK) family. The GroEL (Hsp60) interacts with at least 250 proteins whereby 13 are essential (186). Thus, GroEL is essential at all temperatures. In E. coli, two alternative sigma factors control the heat shock: RpoH (σH) and RpoE (σE). RpoH responds to intracellular stress and is the master regulator of heat shock. RpoE responds to extracytoplasmic stress. At elevated temperatures RpoE regulates RpoH as well as DegP expression. DegP is a periplasmatic endopeptidase essential for growth at high temperatures. (97, 152, 294, 365)

3.2 Protein aggregate disaggregation and protein folding

Newly synthesized peptides, pre-destined to fold to their native structures, are prone to aggregation. Intramolecular interactions define the native protein structure and also lead to aggregated, missfolded proteins. (92) For a protein to carry out its function, a proper fold is needed. In humans, protein aggregates have been connected to various diseases such as the neurodegenerative Alzheimer's and Parkinson's diseases (292). This reinforces the need for protein quality control and disaggregation of protein aggregates. As previously mentioned, bacteria have evolved molecular

chaperone systems that work to avoid protein aggregation and ensure appropriate protein fold. In Escherichia coli four main chaperone systems have been reported: (i) the ribosome-associated trigger factor that works on folding of newly synthesized growing chains. (ii) The Hsp60 system consisting of GroEL (Hsp60) and the cofactor GroES (Hsp10). This system acts on exposed hydrophobic surfaces of compactly folded intermediates. (iii) The third system is the heat shock protein Hsp100 family made up by the Clp ATPases. (iv) The last system is the Hsp70 system, which consists of DnaK (Hsp70), the co-factor DnaJ (Hsp40) and the nucleotide exchange factor (GrpE). The Hsp70 system recognizes polypeptides in an extended conformation. (131, 210, 221, 297, 306, 363, 364) All systems work in an orchestrated manner to supply the bacterium properly folded proteins.

Figure 5. The DnaK/DnaJ/GrpE chaperone cycle system. DnaJ binds and delivers substrate to DnaK. DnaK alters conformation and ATP is hydrolyzed to ADP. GrpE exchanges ADP for ATP, the substrate is released and the cycle is re-charged. Figure adopted from (126).

3.2.1 DnaK/DnaJ/GrpE

Prokaryotes have rather small proteins and it is estimated that 65–80% of them fold immediately after synthesis and that the Hsp70 chaperone system interacts with the rest of the polypeptides (88, 346). The DnaK/DnaJ/GrpE chaperone system is activated upon exposure to high temperature. They assist in a wide variety of cellular processes such as: DNA and RNA synthesis, ribosome assembly, folding of nascent proteins, proteolysis and regulation of the heat shock response (11, 46, 136, 162, 184, 209, 309, 333, 334, 339). DnaK exists in two forms in the cell; a low affinity and a high

affinity state. In its low affinity state, ATP is bound to DnaK and DnaK exhibits low affinity for substrates. DnaJ binds to segments of an approximate eight residues, whereas DnaK prefers hydrophobic cores of four or five residues in length (298, 299). The, by the co-chaperone DnaJ, delivery of substrate to DnaK induces a conformational change in the ATPase domain of DnaK, stimulation of ATP hydrolysis and closing of the DnaK substrate binding cavity. DnaK now has high substrate affinity and low substrate exchange rates. To reset DnaK to an ATP bound state and release the substrate, the nucleotide exchange factor (NEF) GrpE assists. GrpE interacts with DnaK which releases ADP, DnaK is again bound to ATP, and the cycle is reset. (126) (Figure 5)

Figure 6. Disaggregation of protein aggregates by ClpB. ClpB disaggregates proteins from large aggregates in an ATP dependent manner. Medium sized aggregates may be immediately refolded by the DnaK/DnaJ/GrpE system. ClpB may resolubilize small aggregates by a capture and release mechanism or thread substrates through its translocation pore to later refold the substrate with help from the DnaK co-chaperone system. Figure adopted from (203).

3.2.2 The ATPase ClpB

The Hsp100 protein ClpB belongs to the group of AAA+ (ATPase associated with various cellular activities) ATPases (208). In low protein concentration (~0.07 mg/ml) ClpB takes on a monomeric form and at higher protein concentration (>4 mg/ml) an oligomeric, hexameric form dominates (401). The presence of ATP regulates oligomerization of proteins that belong to the Hsp100 family, including ClpB (187, 188, 223, 260). Thermal stress activates ClpB and peptides that are enriched in basic and aromatic residues bind to

ClpB, whereas negatively charged residues (glutamate and aspartate) are disfavored by ClpB (307). Once protein aggregates have formed, chaperone systems like DnaK/DnaJ/GrpE cannot disaggregate the proteins. Thermal stress generates stress-damaged proteins and such proteins tend to aggregate and ClpB has the ability to dissociate these protein aggregates. In an ATP dependent manner large aggregates are turned into medium sized aggregates. The processed aggregates may immediately be refolded by the DnaK/DnaJ/GrpE chaperone system. Other possible scenarios are that the medium sized aggregatess may be resolubilized by ClpB in a capture and release manner or threaded through its central pore (translocation) followed by the refolding step. (Figure 6) (203)

4. Antibiotics and resistance

Human health has benefited greatly from the discovery of antibiotics and the discovery is regarded one of the key accomplishments of modern medicine. The emerging population of multi-resistant bacteria point to infectious diseases that are hard to treat. Many bacteria associated with human diseases have developed substantial resistance to commonly used antibiotics. Examples of multi drug resistant bacteria are: Staphylococcus aureus, Mycobacterium tuberculosis and Streptococcus pneumonia (224). These bacteria are also referred to as superbugs. Depending on their targets or by their effect, antibiotics can be grouped. Bacteriostatic antibiotics (Examples: phenicols, sulfonamides and tetracyclines) inhibit cell growth, whereas bactericidal antibiotics (Examples: cationic peptides/lipopeptides, quinolones and β-lactams) have a more radical effect, namely cell death. Antibiotic resistance is not restricted to clinically isolated bacteria. In the nature many bacteria are naturally resistant to antibiotics and antibacterial compounds are frequently found as naturally occurring products in the environment (20). For instance, Actinobacteria are natural producers of bioactive compounds. The bacterium Streptomyces produces a well known bactericidal aminoglycoside antibiotic, Streptomycin (174, 362). Streptomyces is also resistant to numerous of the currently used antibiotics (76). Alexander Fleming discovered penicillin in 1928 and shortly after this discovery the penicillin inactivating enzyme penicillinase was identified (2, 193). Antibiotics loose effectiveness and new antibacterial substances are needed.

4.1 Antibiotic action and resistance to antibiotics

Existing antibiotics all target common bacterial pathways and structures (Figure 7). Among the targets are: translation, transcription, DNA replication, and cell wall synthesis. Antibiotics can be grouped into various

classes and each class targets different cellular functions within the bacterium (Table 5). As many as the targets, several are the ways the bacteria utilize to overcome the actions by the antibiotic. Examples of how a bacterium can develop resistance to an antibiotic is by introduction of resistance genes into the bacterial genome, alteration of the target structure of the antibiotic, alter porin expression to limit antibiotic uptake and acquisition of efflux pumps that work to lower the intrabacterial concentration of the antibiotic (78, 349). A combination of mutations is many times needed to achieve a high level of resistance. Often an initial mutation offers resistance to the antibiotic and a compensatory mutation (intra- or intergenic) offers fitness compensation needed due to the initial mutation. (218, 254, 344) The intertwined contact between animals and humans has lead to spread of bacteria that have acquired antibiotic resistance from for instance farms. In farms, antibiotics are commonly used not only to treat or prevent bacterial infections, but as a means to assure heavy weight of the animals. (380) It is generally believed that the use of antibiotics in such environments has led to the development of for instance vancomycin resistant Staphylococcus aureus (VRSA) and enterococci (VRE). These emerged resistant bacteria also have implications for the clinic. (23, 37, 202, 332) The appearance of VRSA is thought to have arisen from the use of a vancomycin derivative, avoparcin, and resistance may have been acquired from soil bacteria (350).

Figure 7. Common targets of antibiotics in the bacterium. For more in detail information see (192).

4.2 Bacterial protein translation and the ribosome

In bacteria, transcription and translation occur in a coupled event which means that translation initiates before transcription has ended. Antibiotics have proven to be very helpful tools in determining ribosome structure and function (345). The bacterial ribosome is a complex structure and the translational event carried out by it is greatly organized. The 70S bacterial ribosome is composed of two subunits and more than 50 proteins. The large 50S subunit contains 23S and 5S rRNA (ribosomal RNA) and more than 30

proteins, whereas the small 30S subunit consists of 16S rRNA and 20 proteins. (382) Translation can be divided into four steps: initiation, elongation, termination and ribosome recycling. In the interface of the two ribosomal subunits are three sites employed during translation: the aminoacyl site (A site), the peptidyl site (P site) and the exit site (E site) (395). Figure 8 shows a simplified illustration of the ribosome in translation action. Initiation occurs as the Shine-Dalgarno (SD) sequence of the mRNA interacts with the anti-SD at the 3’ end of the 16S rRNA (139, 317). Initiation requires three initiation factors: IF1, IF2 and IF3 (35). The initiation factors assure correct localization of the start codon in the P site of the 30S subunit and aid in 50S/30S dissociation before initiation (320). IF3 associates with the 30S subunit and prevents association with the 50S subunit and helps to select the methionyl initiator tRNA (fMet-tRNA) and destabilizes other tRNA interaction at the P site (145, 320).

Table 5. Antibiotics, their targets, and resistance mechanisms towards the antibiotics. Adopted from (78).

Class of antibiotic Target Resistance mechanism

β-Lactams/Glycopeptides Peptidoglycan Hydrolysis, efflux, altered target/ biosynthesis Reprogramming of peptidoglycan biosynthesis Aminoglycosides/ Tetracyclines/ Translation Phosphorylation, acetylation, Macrolides/ Phenicols/ nucleotidylation / Monooxygenation / Lincosamides/ Streptogramins/ Hydrolysis, glycosylation, Oxazolidinones phosphorylation/ Acetylation/ Nucleotidylation/ C-O lyase (type B streptogramins), acetylation (type A streptogramins/ Efflux, altered target Rifamycins Transcription ADP-ribosylation, efflux, altered target

Cationic peptides/ Lipopeptides Cell membrane Efflux, altered target

Sulfonamides/ Pyrimidines C1 metabolism Efflux, altered target

Quinolones DNA replication Acetylation, efflux, altered target

IF2 is a GTPase that selectively binds fMet-tRNA (361). IF1 increases IF2 and IF3 affinity to the ribosome (272, 378). This initiator complex with the P site occupied by initiator tRNA joins the 50S subunit and the IFs are released (320, 321). As IF2 is released GTP is hydrolyzed to GDP (137). Now, elongation is initiated. The empty A site receives the by the second codon encoded matching tRNA along with the elongation factor Tu (EF-Tu) and GTP (178). In a complex process, EF-Tu-GTP is hydrolyzed, EF-Tu releases the aminoacyl end to the A site tRNA, which swings into the P site. Now the

P and the A site tRNAs are in the peptidyl transferase site of the 50S subunit and joined by peptide bond formation. (259) The deacylated tRNA from the P site enters the E site and is ejected from the ribosome (284). This translocation event is complicated, but the net outcome is a ribosome ready for the next round of elongation. Termination of translation is determined by the entry of a stop codon in the A site. Bacteria have three stop codons: UAA, UAG and UGA. These stop codons are recognized by release factors (RFs). RF1 recognizes stop codons UAA/UAG, and RF2 recognizes UAA/UGA. (311) The association of the correct RF to the ribosome results in hydrolysis and release of the peptide from the P site (190). Then a third release factor is recruited, the GTPase RF3, which acts to release RF1/RF2 from the A site (113). Release of the peptide leaves the ribosome bound to the mRNA and P site occupied by deacylated tRNA. Complete recycling of the ribosome is generated by the ribosome recycling factor and elongation factor G (EF-G), that together promote dissociation of the ribosome subunits (189). In the end IF3 fulfills its second function; replaces the deacylated tRNA on the 30S subunit and detaches the mRNA, or assists in new SD-anti-SD interaction (179).

Figure 8. The 70S bacterial ribosome in translation mode. The illustration shows a simplified translation event with the 30S and 50S subunits, the associated mRNA and tRNA and a growing polypeptide. The A, P and E sites designate the sites for tRNA entry, peptide bond formation, and exit.

Translation initiation is in general believed to be the rate-limiting step in protein synthesis (178). RF1 and RF2 contain a universal motif consisting of amino acids Glycine-Glycine-Glutamine (GGQ) (91, 115, 148, 304). The motif is thought to enable contact between the RF and the peptidyl transferase center of the 50S ribosomal subunit, but is not essential for RF function (147). Glutamine of the motif is methylated by an S-adenosyl-methionine (SAM) dependent methyltransferase (MTase), HemK. HemK posttranslationally modifies glutamine to N5-glutamine and this methylation stimulates peptide release. (147, 148, 245) Replacement of the second glycine of the motif with an alanine results in a significant slow-down of peptide release from the ribosome (398).

4.3 The T3SS as a target of antimicrobial compounds

The classical method for antibacterial drug discovery was the whole-cell screening (38). The whole-cell screening method has the major drawback in the problematic of finding the target of the drug (133, 227). The method for drug identification later took a new turn to high-throughput screenings (HTSs); cell-free assays that test large amount of compounds (224). HTS requires target identification before screening from compound libraries can be initiated (227, 235). Later, genome sequencing and knowledge of protein structures has opened up the field of antibacterial drug discovery. Other means to attack the topic of antibiotic target identification is to aim for known bacterial virulence markers. These may be inhibition of toxins, targeting toxin delivery mechanisms or affecting regulation of virulence expression (66). Such studies have been conducted in Yersinia, Shigella, Chlamydia, Salmonella, Pseudomonas aeruginosa and pathogenic E. coli and aim to target the T3SS. Acylated hydrazones of different salicylaldehydes have been identified to target the Yersinia T3SS. One compound in particular showed the ability to target T3SS specific Yop secretion. (181, 182, 253) In Shigella, small-molecule T3SS inhibitors affect needle length and secretion system completion (368). Small inhibitory molecules affect various processes in Chlamydia species. Examples of affected functions are: intracellular replication and infectivity, interference with the life cycle and block of effector translocation (19, 242, 243, 383). SPI-1 dependent secretion and invasion can be targeted by small-molecule inhibitors in Salmonella (159). Inhibitors that work on effector translocation have been identified for both Pseudomonas and Yersinia (143). Compounds that target virulence gene expression have been found for pathogenic E. coli (124, 352).

4.4 Virulence-associated genes (Vag) as potential targets for antimicrobial therapy

A recent study set out to identify novel chromosomally located virulence associated genes (vags) through a genome comparison study of hypothetical genes (122). In a bioinformatics approach, genomes of six human pathogens (Treponema pallidum, Borrelia burgdorferi, Helicobacter pylori, Neisseria gonorrhoeae, Streptococcus pneumoniae, and Yersinia pestis) were analyzed and hypothetical genes were limited to the ones annotated in the condensed genome of Treponema palladium. 17 genes were found and analyzed through mutagenesis. 14 of the genes could be mutated, which indicated that the remaining three are essential for survival in vitro. 9 gene mutations of the 14 resulted in attenuated strains in the mouse model. The focus of the study was to identify genes essential in vivo, but not in vitro, therefore these three genes were excluded from the study. After further investigation, 5

mutants remained. Of the initial 14 genes, one gene denoted vagH showed major defects in Yop expression and secretion, but was able to induce a delayed cytotoxic response on HeLa cells. Oral infection of C57BL/6J female mice further showed that the vagH mutant is attenuated in mouse infections. (122) The importance of VagH in vivo, but not in vitro, makes it suitable as a potential target for antimicrobial therapy.

AIMS

The aims of the thesis were to:

 Investigate the specific role of YscU and YscU processing in Yersinia type III secretion system regulation.

 Elucidate the possible contribution of the heat chock chaperone system DnaK/DnaJ in Yersinia T3SS regulation.

 Characterize the potential antimicrobial drug target virulence-associated gene, vagH, and investigate its role in virulence.

RESULTS AND DISCUSSION

5. YscU and its autoproteolytic ability – On the connection to T3SS regulation (Paper I)

Along with YscP, YscU is assigned a role in a sensory process that senses completion of the T3SS needle structure. YscP is a T3SS component found both intrabacterially as well as secreted (264, 330). The existing knowledge on YscU/YscP regulation of the T3SS has mainly been gathered from studies of protein constructs expressed in trans in deletion backgrounds. In this context, YscP shows implications in needle length regulation and Yop secretion initiation and second site mutations in YscU reverse the yscP phenotype (95). YscU is a core component of the T3SS and is crucial for a functional T3SS (12). In addition, YscU belongs to a family of greatly conserved T3SS proteins (160). YscU, as well as YscU homologues in other bacteria, contain a motif consisting of amino acids Asparagine-Proline- Threonine-Histidine (NPTH) (214, 232, 397). This motif confers cleavage ability specifically at position proline, which can be monitored by the appearance of the cleaved product YscUCC. An illustration of YscU and its sub-domains can be found in Paper I, figure 1A. Overexpression of uncleavable YscU variants have been shown fatal. Furthermore, the same study showed no correlation between YscU cleavage and T3S. (201) Later, a second function has been assigned YscU and autoproteolysis. The role as a translocator protein recognizer and subsequent secretion regulator (327).

5.1 YscU is autoproteolytically cleaved in the NPTH motif

In an attempt to illustrate the autoproteolytic processing of YscU, the cytoplasmic portion of YscU (YscUC) was His-tagged and purified in denaturing conditions (8M Urea) from inclusion bodies produced in E. coli.

The on-column purified fraction showed YscUC and its after cleavage interacting products (YscUCN and YscUCC) (Paper I, figure 1B). The purified fraction was diluted to lower urea concentration and left at 21°C overnight.

Subsequent analysis showed an increased pool of YscUCC and YscUCN and less YscUC, which showed that autoproteolysis had occurred (Paper I, figure 1B). N-terminal sequencing confirmed cleavage at position Proline-264.

5.2 Disturbed autoproteolysis affects regulation of T3SS expression and secretion

Analysis of YscU expression and autoproteolysis in the wild-type background showed no correlation between calcium and processing, but expression was

elevated upon calcium depletion, much like the expression pattern of the Yop proteins (Paper I, figure 2). Thus, it is highly unlikely that processing of YscU is the signal for Yop secretion onset. Mutations in the NPTH motif were inserted on the virulence plasmid, in cis. In a mutant where the entire NPTH motif was deleted, YscU was not cleaved and the mutant expressed very low levels of Yops and was unable to secrete them (Paper I, figure 3 and figure 4a). In contradiction to earlier published data the asparagine exchange to alanine (N263A) in the NPTH motif gave rise to moderate YscU autoproteolysis (Paper I, figure 3) (281, 327). Exchange of the proline to alanine (P264A) completely abolished autoproteolysis, whereas threonine could be exchanged to alanine (T265A) without effects on either processing or Yop pattern as compared to the wild type (Paper I, figure 3 and data not shown). A closer analysis of Yop expression and secretion of the mutants revealed regulatory changes in the N263A and the P264A mutants (Paper I, figure 4a-c). Both mutants were derepressed for Yop secretion in +Ca2+ conditions, and also allowed secretion of the negative regulatory protein LcrQ despite calcium concentration (Paper I, figure 4a and figure 6). LcrQ secretion is tightly linked to Yop expression regulation. In the wild type, LcrQ is contained in the bacterial cytosol in non-secretion conditions and released from the bacterium upon calcium depletion (267). While both the N263A and the P264A mutant mutants secrete Yops regardless calcium concentration in the growth medium, secretion was severely reduced compared to the wild type (Paper I, figure 4a-c). Mutations N263A and P264A did not specifically affect Yop secretion, expression of Yops was also affected (Paper I, figure 4c, upper panel). Conformational changes are detectable between processed and non-processed YscU, and it cannot be ruled out that these changes allow only partial secretion blockage, as seen for the leaky N263A and P264A mutants (215). Altogether, this indicated that YscU autoproteolysis is not a requirement for Yop secretion, but it is important for T3SS regulation.

5.3 Proper YscU function is needed for needle regulation

The substrate specificity switch model describes a regulatory model in which YscU and YscP act to jointly regulate the T3SS needle made up by YscF monomers. In this model second site mutations introduced in YscU can suppress the yscP-null mutant phenotype. (95) The yscU-null mutant and the ΔNPTH mutant failed to export needle protein (Data not shown). In calcium containing conditions both the N263A and the P264A mutants displayed elevated levels of YscF compared to the wild type (Paper I, figure 7a). Both mutants showed induced YscF export upon calcium depletion, still in these conditions lower levels of YscF were exported compared to the wild type (Paper I, figure 7a). The regulatory differences seen in the proteolysis

abolished mutants were on the other hand not a result of more fragile needles (Paper I, figure 7b). Interestingly, a closer inspection the yscP mutant showed YscF export levels and pattern much alike the one of the N263A mutant (Paper I, figure 7a). Accordingly, YscU autoproteolysis and YscP are needed for proper regulation of the needle.

5.4 Motif mutants assemble functional T3SS machines

Closer analysis of secretion by the motif mutants N263A and P264A clearly illustrated secretion of all translocator proteins (YopB, YopD and LcrV), albeit at lower levels than the ones seen for the wild type (Paper I, figure 4a-c and figure 5). This contradicts findings in a previous study where it was described that YscU autoproteolysis is responsible for translocator protein secretion (327). Yersinia applied onto HeLa cell monolayers are able to induce a rapid cytotoxic response caused by the translocation of the cytotoxin YopE. This is monitored as altered cell morphology, and more precisely cell rounding. (288-290) Secretion of translocator proteins is an absolute requirement for effector translocation (164, 266, 290). Mutants N263A and P264A were both able to induce a delayed cytotoxic response on HeLa cells (Paper I, figure 8). This indicates that mutant YscU protein abolished in autoprocessing still assemble a functional T3SS and that YscU does not act as a discriminator that sorts effectors from translocators. On the other hand, YscU appears to have a more general role in control of the T3SS and secretion by it.

5.5 Proposed model of YscU action

The phenotypic characterization of in cis constructed YscU motif mutants abolished of autoproteolysis emphasizes the importance of YscU cleavage for negative regulation of Yop secretion and YscF export in T3SS non-inducing conditions. Such are the findings that they provide a minor change to the proposed regulation originally described in the substrate specificity switch model of the Salmonella flagellum. This model describes how polymerization of the hook protein, FlgE, stops to the benefit of filament, FliC, assembly (377). The hook in the Yersinia Ysc/Yop T3SS corresponds to the needle (YscF) and the filament to the Yops. The results in Paper I propose a parallel secretion of YscF and Yops, where autoproteolysis is required for negative regulation of secretion of both substrates in plus calcium conditions.

Figure 9. Model of YscU autoproteolysis regulation of the T3SS. A. In the wild type. Prior to target cell contact (+Ca2+ conditions) YscU is processed, and processing is needed for negative regulation of Yop/YscF secretion. After autoproteolysis processed YscUCC remains in close contact with YscUCN. Upon target cell contact the by processing added negative block in secretion is relieved. YscUCC is dissociated from YscUCN and this act of relief may be provided by YscP. B. The N263A/P264A mutants. YscU cannot undergo autoproteolysis and adopts an altered conformation. Yops may leak prior to target cell contact (+Ca2+ conditions), and YscF is constitutively secreted. Upon target cell contact YscP is unable to dissociate YscUCC from YscUCN and target cell contact is not sensed properly.

The proposed modified model (Figure 9) includes that correct autoproteolysis of YscU relieves the block in YscF and Yop release made by

non-processed YscU. This model may appear contradictory due to the finding that non-processed YscU allows YscF and Yop secretion in +Ca2+ conditions. Furthermore, there was no correlation between calcium and processing (Paper I, figure 2). YscU expression is induced upon calcium depletion and YscUCC and YscUCN interact after cleavage (Paper I, figure 1b and 2). It is possible to envision that processing is not enough to separate the two cleaved YscU parts from each other and that this event is required for proper YscU function. Accordingly, processing of YscU is essential to produce a block in Yop secretion and this block is relieved upon target cell contact in vivo or calcium depletion in vitro. The N263A mutant and the yscP-null mutant showed similar YscF export pattern. Furthermore, the extragenic suppressor mutations that reverse the yscP mutant phenotype and allow for Yop secretion of the yscP mutant are all located in YscUCC. This makes YscP a potential candidate for the YscUCC/YscUCN subunit separation. The regulatory phenotypes seen for mutants N263A/P264A are most likely the result of faulty displacement of YscUCC due to altered conformation that only in part can block Yop secretion.

6. DnaK/DnaJ are needed for proper T3SS function (Paper II)

The heat shock chaperone proteins DnaK and DnaJ work in an orchestrated manner to assist in many cellular functions. Among these are: DNA and RNA synthesis, ribosome assembly, folding of nascent proteins, proteolysis and regulation of the heat shock response (11, 46, 136, 162, 184, 209, 309, 333, 334, 339). Stress conditions activate the DnaK/DnaJ system along with important proteases and AAA+ ATPases. It has been hypothesized that the DnaK/DnaJ system allows for survival of facultative intracellular pathogenic bacteria (Yersinia enterocolitica, Legionella pneumophila, Salmonella enterica serovar Typhimurium) within the macrophage phagosome after phagocytosis. The idea is based on the finding that the heat shock system is induced as the pathogens entered the macrophage. (3, 43, 391) The DnaK/DnaJ machinery also provides Salmonella epithelial cell invasion capacity, secretion capacity of SPI-1 encoded proteins (SipA, SipC and SipD) and flagellar proteins, as well as the ability to survive within macrophages (340). Furthermore, mutations in dnaK and dnaJ result in non-motile bacteria (315).

6.1 DnaK/DnaJ are needed for a functional flagellar T3SS and Yop T3SS

The DnaK/DnaJ system is involved in a variety of cellular processes which explains why lack of these proteins lead to pleiotropic effects. In this study,

both genes were deleted by insertion mutagenesis in Yersinia and motility was monitored on soft agar plates. In E. coli, mutations of dnaK/dnaJ lead to loss of motility, and this is also the case in Yersinia (Paper II, figure 2). Next, the mutants were tested for their ability to induce a cytotoxic response on HeLa cells, a phenotype caused by the YopE protein. Compared to the wild type, which induced a rapid cytotoxic response, both the dnaJ mutant and the double dnaK/dnaJ mutant were severely defective in their ability to induce a response on HeLa cells (Paper II, figure 1). In conclusion DnaJ and DnaK are needed both for flagellar driven motility and for a fully functional Ysc/Yop T3SS competent of Yop translocation into HeLa cells.

6.2 The connection between the HSPs and T3SS regulation

The cytotoxicity assay on HeLa is a measure of T3SS functionality and a delayed or abolished cytotoxic response indicates T3SS regulatory defects. To our surprise, the dnaJ mutant had not lost Yop secretion regulation and showed Yop secretion like the one of the wild type (Paper II, figure 3). The dnaK/J mutant also displayed wild-type like Yop regulation control, but secreted less than the wild type in -Ca2+ growth medium (Paper II, figure 3). These findings are the complete opposite of the ones reported for Salmonella, which was greatly unexpected. The results place both HSPs in a Yop secretion and expression regulatory role. On the contrary, both hsp- mutants displayed deregulated needle export to the bacterial surface in +Ca2+ conditions (Paper II, figure 4). Upon calcium depletion the dnaJ mutant exported YscF at wild-type levels, whereas the dnaK/J mutant showed lower YscF export (Paper II, figure 4). The derepressed YscF phenotype seen in the mutants greatly resembled the one of the autoproteolysis defective N263A and P264A mutants in the YscU NPTH motif (Paper I, figure 7). Indeed, YscU autoproteolysis was also affected in the hsp-mutants (Paper II, figure 5). This argues for a role of the HSPs in YscU autoproteolysis. More importantly, the in vitro Yop secretion can be uncoupled from in vivo Yop translocation and this uncoupling can be correlated to the HSPs. It is likely that the HSPs have a direct role in substrate specificity switching from YscF (needle protein) to Yop secretion/translocation in vivo.

6.3 Full-length YscU associates with the bacterial OM

YscU has successfully been described as a protein inserted in the inner membrane (12). To determine if YscU unable to undergo autoproteolysis still localized to the IM, bacterial total membranes were separated by sucrose gradient centrifugation. Remarkably, YscU in its full-length form showed association with the outer membrane (Paper II, figure 6). This OM

association was seen for mutants N263A, P264A and for both hsp-mutants.

YscUCC was found still associated with the IM (Paper II, figure 6). YscUCC localization to the IM supports our previous finding that YscUCC and YscUCN associate after autoproteolysis (Paper I, figure 1B). OM fractions containing

YscUFL were subjected to gentle detergent wash and this released the YscU protein (Paper II, figure 7). This shows that YscUFL is associated with, but not integrated in the OM, but likely associates with components of the T3SS present in the OM. It cannot be ruled out that YscU, perhaps through its periplasmic loops present in YscUTMS, has a function in T3SS gating through interaction with OM components.

6.4 DnaJ specifically interacts with defined YscU domains

The effect seen on YscU autoproteolysis prompted HSP/YscU interaction studies. GST-fusion proteins were made and used in co-precipitation studies. It was found that YscU interacts specifically with DnaJ and the DnaJ binding domain (DnaJBD), but not with DnaK or its binding domain and not with the general protein folding chaperone GroEL (Paper II, figure 8A). To further dissect which parts of YscU that constituted this interaction, the different domains of YscU (YscUFL, YscUTMS, YscUC, YscUCN and YscUCC) were fused to GST and individually analyzed for DnaJ co-precipitation (Paper II, figure 8B and 8D). Interactions were found between DnaJ and full-length YscU,

YscUTMS, YscUC and YscUCN (Paper II, figure 8B). The strongest interaction was detected with YscUCN. YscUCN/DnaJ interaction was once again verified on peptide mapping interaction studies, where interaction also was identified in the one and only YscU cytosolic loop of the YscUTMS domain. The interaction studies point out distinct areas of YscU where interaction occurs with DnaJ.

6.5 Model of DnaK/DnaJ T3SS regulation contribution

The HSP-system works on many cellular processes, whereby one is to ensure proper protein folding. The incapability of YscU autoproteolysis in the hsp- mutants may be a result of improper YscU autoproteolysis due to the lack of HSP folding assistance. Another attractive idea is that the HSPs act specifically as a part of the T3SS. DnaJ interaction to YscUCN may be a prerequisite for proper T3SS regulation and secretion. For this activity interaction to occur, YscUCC displacement from YscUCN may be required.

Figure 10. DnaK/DnaJ and the T3SS. A. Before target cell contact (+Ca2+) wild-type YscU adopts a conformation needed for negative Yop and YscF secretion regulation. Upon calcium depletion, or target cell contact, YscP may displace YscUCC from YscUCN. DnaJ may be needed for proper YscU fold or as an active part of the T3SS by binding to YscUTMS and YscUCN. B. In dnaK/dnaJ mutants YscU is improperly folded. This allows constitutive YscF secretion, but keeps Yop secretion regulation. Target cell contact (-Ca2+) is not sensed by the T3SS and Yops are secreted but translocation is poor.

Correct autoproteolysis of YscU is needed to place negative regulation on Yop secretion and T3SS induction, and either target cell contact or calcium depletion, relieves this block (Paper I and figure 9 in this thesis). Depletion of the hsp genes may give rise to an improperly folded YscU, unable to undergo autoproteolysis and correctly regulate the needle, but still capable of proper Yop secretion regulation in vitro (calcium depleted media). To

conclude, the HSPs and proper YscU autoproteolysis are needed to accurately regulate the T3SS in response to target cell contact (or calcium depletion). Figure 10 shows a simplified illustration of HSPs and their proposed function T3SS regulation.

7. VagH and the link to the T3SS and virulence (Paper III)

VagH shows homology to E. coli HemK. HemK was first identified as a protoporphyrinogen oxidase and later thought to be a DNA methyltransferase before the correct function as a glutamine methyltransferase was established (45, 147, 245, 248). In E. coli, the MTase activity of HemK targets RF1 and RF2 (147, 245). RFs are recruited to the translation site as the ribosome encounters a stop codon in the translated mRNA. The role of the RFs is to induce release of the synthesized peptide. A conserved GGQ motif in RFs from all kingdoms has been identified and the glutamine in this motif is posttranslationally modified to an N5- methylglutamine (91, 115). Glutamine methylation is very rare and only one more bacterial protein has been shown to undergo such modification: the ribosomal protein L3 (68, 207, 271). In E. coli K-12, a hemK mutant shows severe growth defects (147, 245). This growth defect can be in part restored by adding an additional mutation in RF2, where the tyrosine at position 246 is replaced by either a serine or an alanine (245). The tyrosine makes the RF2 of E. coli K-12 more dependent on methylation for efficient translational termination (358). Most other bacterial RF2s encode an alanine or a serine in the corresponding position which likely make them less dependent on methyl transfer for efficient termination. A mutant in vagH of Yersinia pseudotuberculosis does not display growth defects in rich growth medium (122).

7.1 VagH has methyltransferase activity and targets RF1 and RF2

HemK and VagH share homology (63% identity), which suggested that VagH also may have methyltransferase activity. To identify whether VagH also had MTase action, protein lysates of Yersinia were incubated with purified VagH 3 3 and radio-labeled SAM, [ H]SAM (67). Incorporation of [ H]CH3 was evaluated by autoradiography or by scintillation counting. The results show that in the wild-type lysate the targets are saturated, and like with HemK of E. coli, RF1 and RF2 are methylated by VagH (Paper III, figure 1-3). It was further observed that Yersinia RF2 had different migratory capacity on SDS- PAGE than E. coli RF2 (Paper III, figure 3). The theoretical molecular weight of these two proteins is very similar (42 Da difference), which makes understanding of altered migration difficult to comprehend.

7.2 Microarray and proteomics - VagH in the scene of T3SS regulation

Microarray analysis was performed on the vagH mutant and the wild type. mRNA was prepared from both strains, and the corresponding cDNA hybridized to a DNA chip containing the genes that encode all predicted ORFs of Y. pestis. In addition, bacterial lysates of the same strains were prepared and protein spots from 2D gel electrophoresis were analyzed for up- or down-regulated proteins in the mutant compared to the wild type. In the E. coli hemK mutant a microarray analysis showed 260 genes differently expressed than in the wild type (245). Surprisingly, the vagH mutant only showed 22 genes differently regulated, whereby six were down-regulated ribosomal proteins (Paper III, table 1). No identified gene was obviously involved in virulence. The finding that ribosomal proteins were affected in regulation pointed to translational effects due to vagH deletion rather than transcriptional. The proteomics study also identified very few proteins differently expressed in the two strains and five proteins were identified by mass spectrometry (Paper III, table 2). One of the proteins was found two- fold increased in the vagH mutant: the protein YopD. YopD is a T3SS negative regulator, and the up-regulation of YopD correlates well with the observed down-regulation of the T3SS in the vagH mutant (Paper III, figure 5) (111, 122, 376). The down-regulation of T3SS Yop expression and secretion in the vagH mutant was further confirmed and the negative regulation conferred by YopD could be relieved by deletion of yopD in the vagH mutant (Paper III, figure 6). To determine if the MTase activity of VagH is important for T3SS regulation, an enzymatically inactive VagH protein and wild-type VagH were expressed in the vagH mutant. Wild-type VagH could complement the mutant, but the enzymatically inactive VagH could not (Paper III, figure 5). Another interesting protein that appeared from the proteomics analysis was a close to three-fold down-regulated hypothetical iron-binding protein. Iron acquisition within the host is important for virulence, and down-regulation of an iron-binding protein could explain the virulence attenuation of the vagH mutant (44). The gene encoding the protein was deleted and mice were orally infected by the mutant. The mutant was as infectious as the wild type. All further phenotypical analyses of the mutant have unsuccessfully linked the protein to T3SS regulation, iron dependency or susceptibility to macrophage killing or macrophage and HeLa cell intracellular survival and replication (data not shown).

7.3 VagH is needed for virulence and macrophage survival

The vagH mutant was clearly attenuated in the mouse model, delayed in the HeLa cell cytotoxic response, and it had apparent lowered Yop secretion

compared to the wild type (122). Now, virulence was assayed in greater detail. C57BL/6J female mice were infected intraperitoneally and the in- frame deletion vagH mutant was found 500-fold less virulent than the wild type. The vagH mutant was also orally administrated and organs were monitored over time. The vagH mutant behaved much like Yersinia cured of the virulence plasmid and failed to colonize Peyer’s patches and the spleen (Paper III, figure 4). The Yersinia pseudotuberculosis T3SS mainly act to inhibit phagocytosis by macrophages (287). Survival and replication within macrophages may be important for Y. pseudotuberculosis virulence, but is not dependent on the T3SS (135, 276). To exclude factors responsible for the attenuated phenotype other than the T3SS, macrophage intracellular survival was investigated (Paper III, figure 7). To avoid T3SS effects, non- induced vagH mutant and wild-type bacteria were allowed to enter macrophages. After different time points the macrophages were lysed and viable counts were made. Both strains could replicate and survive within the macrophages equally well (Paper III, figure 7A). Macrophages possess the ability to rapidly clear virulence-plasmid-cured Yersinia from the culture (22). To test the survival of the vagH mutant and the wild type in the presence of macrophages, the T3SS was induced (-Ca2+ pre-growth) and bacteria were added to macrophages. This assay showed that after eight hours of infection, the vagH mutant was more efficiently killed by the macrophages than the wild type (Paper III, figure 7B). The same infection strategy applied on nonprofessional phagocyte HeLa cells, on the other hand, showed no difference between the two strains (Paper III, figure 7C). In such a setting, a difference has been seen between the wild type and plasmid cured Yersinia (22). Most likely, the reason why differences were not detected between the vagH mutant and the wild type is that the vagH mutant has a functional T3SS and is able to induce a cytotoxic response on HeLa cells (though delayed) (122). The conclusion is that the effect on the T3SS (lowered Yop secretion), as a consequence of vagH deletion, blocks the ability of the bacteria to inhibit phagocytosis by macrophages.

8. Further remarks

YscU autoproteolysis was earlier shown not to be important for T3S, but for bacterial survival (201). This conclusion was drawn from results collected from mutants with gene constructs expressed on introduced plasmids; in trans. This study and the results from paper I show obvious differences between data collected with plasmids introduced in trans in mutant backgrounds and mutations introduced in cis. Altogether, this clearly highlights the importance of performing studies in their native condition. The reasons why the outcomes of the studies are so diverse may be manifold. Excess YscU provided by multi-copy plasmids and over expression by non-

native promoters may not only cause ill-timed YscU production, it may also cause IM insertion in an environment where it lacks other Ysc machinery components essential for function. When expressed in trans, the levels of YscU were very low in the motif mutant constructs (201). This might be the outcome of stability problems. It cannot be ruled out that YscU needs other T3SS components for its function and stability. Furthermore, the IM insertion ability of YscU, on its own, may be the answer why over expression of YscU in trans is toxic. Another possible explanation is that overexpressed YscU may not adopt a functional fold required for proper T3SS regulation.

This is supported by the fact that when not induced by IPTG, YscUFL and uncleavable YscU variants (N263A and P264A) are able to confer Yop secretion in calcium depleted media. Furthermore, upon closer inspection visibly lower Yop secretion is seen when N263A/P264A YscU are expressed in trans in the yscU mutant. (201) This lowered secretion compared to the wild type fits very well with the findings now presented with in cis introduced N263A/P264A mutations.

CONCLUSIONS

The main findings in this thesis are:

 YscU is autoproteolytically cleaved at position Proline-264 of the conserved NPTH motif. The NPTH motif is crucial for a functional T3SS.

 Autoproteolysis is required for proper expression and secretion of Yop proteins and LcrV.

 Mutations in the NPTH motif that abolish cleavage (N263A and P264A) prematurely leak the negative regulatory protein LcrQ in non-permissive secretion conditions.

 YscU autoproteolysis is important for negative regulation of Yop secretion and YscF export. Target cell contact, or calcium depletion, relieves this negative block.

 DnaJ and DnaK are needed for motility and proper T3SS regulation.

 Mutants in dnaK/dnaJ show deregulated YscF export, wild-type like Yop secretion regulation and significant defects in cytotoxic response on HeLa cells.

 The dnaJ and the dnaK/J mutants, and yscU mutants with abolished

YscU processing, show YscUFL associated with the bacterial outer membrane.

 DnaJ and its binding domain interact with YscU. The interaction occurs

in the YscU cytoplasmic loop and in YscUCN.

 VagH is an S-adenosyl-methionine dependent methyltransferase and targets RF1 and RF2.

 The vagH mutant shows down-regulated T3S and a two-fold increased expression of the negative regulator YopD, it is attenuated in the mouse model, but is not affected in macrophage intracellular survival.

ACKNOWLEDGEMENTS

I have spent nearly eleven years in Umeå, and a lot of them at the department of molecular biology. I have been fortuned to meet many amazing people in my life, and to those I now want to express my gratitude. Hasse. Tack för att du tog dig an mig! Du har en stor portion tålamod och har lyckats hantera min halvfinska, envisa personlighet med bravur. Jag har lärt mig massor av att jobba med dig och du är en utomordentlig vetenskapsman. Present members of the HWW group: Frédéric, you are an amazing person and you deserve more flowers! Stefan you are kind and calm. Keep the lab structurized when I leave. Roland N, tack för allt gott kaffe och HeLa celler. Tomas, Mr. Passion for Science. Kör i vind! Helen, you are gifted and will go far. Good luck with baby Harry. Former members of the HWW group: Elin, du är en klippa och helt oersättbar i mitt liv. Tvivla aldrig på vad du kan! Du är en underbar mamma till finaste Alice och jag är så tacksam att jag har dig som vän! Rara Sara G, jag är evigt tacksam för allt visat tålamod och för allt du har lärt mig. Lycka till med familjen! Margareta, hoppas att du trivs i Uppsala och att din dröm om Australien blir verklighet. Martina, lycka till med familjen. Janne, du är intressant. Sara S. B., den hemliga doktoranden. Du är en fantastisk människa och en fin vän! All other Yersinia people: Katrin, om jag någonsin blir mamma hoppas jag att jag blir en lika bra mamma som du är. Du är en fantastisk människa! Sara T, du har en fin själ! Lycka till med mammalivet. Anna F, krutmorsa med driv och stark motivation. Lycka till med allt! Sofie, snäll som få. Lycka till med din bok. Karen, good luck with everything. Kemal and Ümmühan, good look with your PhD’s. Maria F, kom ihåg att andas och tack för pepp och feedback. Roland R, tack för all vänlighet. Åke F, tack för mutanter och hjälp med YscU. Moa L, tack för fint samarbete och för all YscU hjälp! Journal club and group meetings and seminar people: Matt, thanks for good input. Kristoffer, lycka till med familjen, avhandlingen och the fishy business. Vicky, thank you for all the paper signing prior to my defence and for all your feedback on seminars. Other molbiol people: Sven, var har du muggen och ännu viktigare har du koncernöverblick? Tord, för alla LADOK-dokument som till sist blev rätt. Administrative personnel: Speciellt tack till Helena. Mediapersonal, som underlättat labarbetet avsevärt. All former and present PhD and postdoc colleagues: No one mentioned, no one forgotten. Thank you! The German/Austrian/French/Spanish friends: Lena, guacamole queen. Du är bra fin du! Joanna, I love your sharp tongue and you’re a good baker. Christina, musikanten och äventyrerskan. Snart är det din tur. Fanny, good luck with everything. Benedikt, lycka till med din avhandling. Dominik, good luck. David E, lycka till i söder! Hinnerk and Kerstin,

good luck with baby Annie. Michaela, tuff tjej med mjukt hjärta. Du är snäll! Sophie, best beef tartare ever. Rebekka, living on the edge sometimes causes pain. Watch out for the Umeå effect! Birgit, min juvel. Tack för PicQues, bus, skratt, gråt, resor, ansiktsmasker, kärlek…för att du är du och för att du är min vän! Richard, för att du tar hand om Birgit. Teresa, Miss dancing queen. Tronca, you are amazing and that not only because you put up with my Spanish. Martin T, tack även du för att du tar hand om min vän Teresa. Barbara, I am so happy to call you my friend. Always there, always loving, always caring. I hold a very special place in my heart for you! Willi, tall and kind. I wish all the best for you and Babsi. Andreas Ö/Andi, you are a good person and I wish you all the best. Folk med ursprung från eller anknytning till molbiol: Sofia E, vänligare människa får man leta efter. Tack för att du alltid bryr dig om. Jeanette och David, lycka till med familjen. Maria, se över Tunnelbacken när jag flyttat. Petra E, du är en fin människa. Lycka till med familjen! All my friends in ‘the real world’: Erika L, många års vänskap och jag värdesättar vartenda ett av dom. Du är toppen! Nils F, ingen dricker gin som du. Du och Erika är fina föräldrar och det märks på underbara Ludvig och Selma. Emmsan, godhjärtat och glad. Du värmer mitt hjärta. Anders, du är rolig och så bra för Emma. Jag ser fram emot att hälsa på er i Oslo. Lars, grattis till lilla Alice! Jag är glad att Elin träffade just dig. Andreas T, du kommer att bli en bra pappa. David S, Mikael M, Stephan och Oskar, tack för galna fester, metal och schlager. Lisa, vi ses i Stockholm. Ulrik, fd granne. Tack för alla våfflor. Tommy, schlagerfrälst och go. Viktoria, minst lika schlagertokig och väldigt omtänksam. Terese A, du är en bra Kalixjänta! Mikael S, jag tycker om dig Masken. Andreas H, du skånska virvelvind. Lycka till med familjen och tack för att du är en så fin vän. Johan W, vi ses i Stockholm fina vän! Alla Davids fantastiska vänner som jag har glädjen att också kalla mina: Set och Emma, Rickard och Jenny, Stina och Martin, Johan och Magdalena, Yvonne, Jonas och Helena, Henrik J, Thomas S. Vi ses i Stockholm! Min familj: Familjen är det som står mig närmast hjärtat. Mamma, rakas äiti! Du ställer upp i vått och torrt och jag är så glad att just du är min mamma! Pappa, du är så duktig på att laga mat! Jag uppskattar dig och ditt varma hjärta. Plastmamma Barbro och plastpappa Göran, det är guld värt att ha extra föräldrar. Ni är fina! Bror Anders, du är otroligt kärleksfull och du är en fin människa. Bror du är en klippa! Kul att du träffat Oksana. Bror Niklas och Hanna, ta hand om varandra i Wien. Älskade ovärderliga lillasyster Cecilia, vem vore jag utan dig?! Vi har skrattat och gråtit ihop, delat hemligheter och ingen känner mig som du. Tack för att du alltid finns där för mig! Örjan, tack för peppen med specialarbetet, och tack för att du alltid gör att jag känner mig välkommen i ditt och Cecilias hem. Jag kan inte tänka mig någon annan för min lillasyster! Tack även till resten av min

familj och speciellt tack till moster Ritva för att du fick mig att inse det fina med orkidéer. Tack även till Davids familj som så varmt välkomnat mig. Torsten, Iva och Jenny - David har en fin familj och jag uppskattar er innerligt!

Sist, men absolut inte minst: Min ögonsten och kärlek, David. Jag skulle kunna skriva en hel bok om vad jag känner för dig och hur lycklig jag är av att ha dig i mitt liv. Du kompletterar mig och får mitt hjärta att slå kullerbyttor. Jag är så glad att du har stått ut med att ha mig på distans i nästan två år. Du är så omtänksam, tålmodig och kärleksfull! Jag ser fram emot vår framtid och att äntligen få bo tillsammans och skapa vardag. Du är bäst och jag älskar dig!

This work was performed within the Laboratory for Molecular Infection Medicine Sweden (MIMS) and the Umeå Centre for Microbial Research (UCMR) and was supported by funding from the Swedish Research Council, the UCMR Linnaeus Postdoctoral Program and the JC Kempe Memorial Fund.

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