Molecular mechanisms of Yersinia pseudotuberculosis for adaptation and establishment of infection in host tissue A K M Firoj Mahmud Department of Molecular Biology Umeå Center for Microbial Research (UCMR) Laboratory for Molecular Infection medicine Sweden (MIMS) Umeå University, Umeå 2021 This work is protected by the Swedish Copyright Legislation (Act 1960:729)a Dissertation for PhD ISBN: 978-91-7855-488-1 ISSN: 0346-6612 Revealing bacterial molecular mechanisms Cover page design by: A K M Firoj Mahmud Electronic version available at: http://umu.diva-portal.org/ Printed by: print and Media Umea, Sweden 2021 To my Mother, Sister and Wife, who always keep their faith on me Any living cell carries with it the experience of a billion years of experimentation by its ancestors … Max Ludwig Henning Delbrück (1949) Table of Contents Abstract ............................................................................... i Abbreviations ................................................................. …iii Papers included in this thesis ......................................... …V Background…………..…..…………………….….…………………….......1 1. Introduction. ………………………………………………..…….………..3 1.1 Bacterial evolution from origin to pathogenesis ………………………………………..3 1.2 Host pathogen interaction and adaptability: from bacterial perspective ........ 4 1.2.1 Virulence factors… ....................................................................................... 4 1.2.2 Adherence and colonization factors ............................................. 4 1.2.3 Invasion factors: exoenzymes, toxins and others ...................................... 5 1.3 Stress responses…………………………………………………………………………………….6 1.4 Response to multiple stressors during infection………………………………………..7 1.5 Biofilm formation……. ....................................................................................... 8 1.6 Quorum sensing…….. ...................................................................................... 10 1.7 Bacterial heterogeneity .................................................................................... 11 1.8 Persistent infections… ..................................................................................... 12 2 Molecular mechanisms underlying bacterial adaptation and establishment of infection ..…………………………………………..…13 2.1 Transcription factors and gene regulation ..................................................... 13 2.2 Sigma factors in controlling global gene expression ...................................... 14 2.2.1 The alternative sigma factor RpoN ...........................................................15 2.2.1.1 Role of RpoN in bacterial virulence ............................................... 16 2.3 Post transcriptional regulation and translational control. ............... 17 3 Yersinia pseudotuberculosis as a model organism to study the molecular mechanism of pathogenic bacteria ……………...18 3.1 Yersinia biofilm formation……………………………………………………………………19 3.2 RpoN regulation in Y. pseudotuberculosis ............................. 20 3.3 Yersinia type 3 secretion system (T3SS)................................. 20 3.3.1 The secretion apparatus ...................................................... 21 3.3.2 Effector proteins ................................................................ 22 3.4 Yersinia Infection ................................................................... 24 4. Next generation sequencing to reveal the Molecular mecha- nisms of bacteria………………………………………………………………….26 4.1 RNA-Seq…………………………………………………………………………………………….28 4.2 ChIP-Seq:………………… .................................................................................... 29 4.3 RNA-Seq data analysis: challenges and limitations in prokaryotic research 31 4.3.1 RNA-Seq data processing .......................................................................... 32 4.3.2 Gene expression comparison between conditions .................................. 32 4.3.3 Differential expression .............................................................................. 34 4.3.4 A user-friendly tool is required ................................................................ 35 5. Aim of the study…………………………………………………….…………….…………36 6. Results and discussion……………….…………………………………….……………..37 6.1. ProkSeq, a complete RNA-Seq data analysis package for prokaryote, reduces data handling complexity and ensures reproducibility ................................ 37 6.2.1 Yersinia pseudotuberculosis forms biofilm to adapt to different stress con- ditions…………………….. .................................................................................... 38 6.2.2 Similar regulation of a smaller set of core genes indicates the existence of central but complex gene regulation in biofilm ............................................. 39 6.2.3 Metabolic reprograming in biofilm ............................................................40 6.2.4 RpoN is important for biofilm formation ................................................... 40 6.3.1 Genome wide RpoN Binding sites in Y. pseudotuberculosis, are revealed by ChIP-Seq …………………. .................................................................................. 42 6.3.2 RpoN act as both an activator and repressor ............................................. 43 6.3.3 Intragenic binding sites of RpoN and gene regulations ............................ 44 6.3.4 Cross-talk between sigma factors and RpoN regulation ................... 45 6.4 RpoN plays an important role in virulence and is required for T3SS ........ 46 7. Outcomes and main findings in this thesis ……………..……………………..49 8. Future perspectives …………………………………………………………………….....50 Acknowledgement………………….…………………………………………………………54 References ….…………………………………………………………………………….59 Abstract Bacterial pathogens can evade the host’s immune defence to adapt and establish an infection within the host. Some even slip into a quiescent state to establish themselves without acutely harming the host. Phylo- genetically unrelated bacteria can share similar strategies for the es- tablishment of infection and for persistence. Our lab previously showed that Yersinia pseudotuberculosis underwent a dramatic re- programming from a virulent phenotype expressing virulence genes, including T3SS and Yop effectors during early infection, to an adapted phenotype capable of persisting in tissue. The overall aim of my PhD study was to dissect the mechanisms behind bacterial adaptation and maintenance of infection within host tissue using Y. pseudotuberculo- sis as a model pathogen. The ultimate goal is to identify key players of critical importance for the ability of the bacterium to maintain and es- tablish infection in host tissue. In my studies, I mainly focused on bac- terial biofilm and the role of the alternative sigma factor RpoN. Much of my studies involve RNA-Seq analyses, encouraging me to develop a convenient, time-efficient, and all-purpose RNA-Seq data analysis package especially designed for prokaryotic organisms. The package is available online as a free tool and can be used by any biologist with minimal computational knowledge. We systematically examined bio- film formation of Y. pseudotuberculosis under different stress condi- tions and found that biofilm development involved a series of adaptive responses against various stressors, including bile, pH, amino acid deprivation, and temperature and oxygen-level changes. Analyses of transcription profiles of bacteria forming biofilm in different condi- tions revealed a set of core genes that were similarly regulated in bio- film bacteria independently of induced environment. The transcrip- tional regulator RpoN, commonly known as sigma 54, was found to be important for biofilm formation, and a ∆rpoN mutant strain was se- verely attenuated in virulence. To understand the regulatory mecha- nisms involved, we investigated gene expressions in wild-type (WT) and the isogenic ∆rpoN mutant strain and also chromatin immuno- precipitation followed by sequencing. We have identified RpoN bind- ing sites in the Y. pseudotuberculosis genome and revealed a complex i regulation by RpoN involving both activation and repression effects. We also investigated the role of RpoN in regulation of the Type III se- cretion system (T3SS) and found that RpoN was required for a func- tional T3SS, which is essential for bacterial virulence properties in host tissue. Our work indicates that Yersinia modulates itself in mul- tiple ways to create niches favourable to growth and survival in the host environment. We have identified some key regulators and genes that will be explored further for their potential as novel targets for the development of new antibiotics. ii Abbreviations ATP Adenosine tri phosphate CDSs Coding sequences CFUs Colony forming units DNA Deoxyribonucleic acid EBPs Enhancer-binding proteins ECM Extracellular matrix FPKM Fragment per kilobase per million GO Gene ontology GSEA Gene set enrichment analysis Hms Hemin storage kDa Kilodalton KEGG Kyoto encyclopedia of genes and genomes LPS Lipopolysaccharides MLNs Mesenteric lymph nodes mRNA Messenger ribonucleic acid MDR Multidrug resistant bacteria Nitrogen cycle N-cycle NGS Next-generation sequencing ncRNAs Non coding RNAs PATRIC Pathosystems resource integration center PPs Peyer’s patches QC Quality control QS Quorum sensing RPKM Read per kilobase per million RNA Ribonucleic acid RBSs Ribosomal binding sites rmc RNA molar concentration RNAP RNA polymerase SFs Sigma Factors iii TPM Transcript per million TF Transcription factor TFs Transcription