Regulation of the Locus of Enterocyte Effacement in Attaching and Effacing
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1 Regulation of the Locus of Enterocyte Effacement in Attaching and Effacing 2 Pathogens 3 4 5 R. Christopher D. Furniss, Abigail Clements# 6 7 MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, 8 Imperial College London, London SW7 2AZ, UK. 9 10 #Address correspondence to Abigail Clements, [email protected] 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 1 29 30 Abstract 31 Attaching and Effacing (AE) pathogens colonise the gut mucosa using a Type Three 32 Secretion System (T3SS) and a suite of effector proteins. The Locus of Enterocyte 33 Effacement (LEE) is the defining genetic feature of the AE pathogens, encoding the 34 T3SS and the core effector proteins necessary for pathogenesis. Extensive research 35 has revealed a complex regulatory network that senses and responds to a myriad of 36 host and microbiota-derived signals in the infected gut to control transcription of the 37 LEE. These signals include microbiota-liberated sugars and metabolites in the gut 38 lumen, molecular oxygen at the gut epithelium and host hormones. Recent research 39 has revealed that AE pathogens also perceive physical signals, such as attachment 40 to the epithelium, and that the act of effector translocation remodels gene expression 41 in infecting bacteria. In this review we summarise our knowledge to date and present 42 an integrated view of how chemical, geographical and physical cues regulate the 43 virulence program of AE pathogens during infection. 44 45 46 Introduction 47 48 Initially described by the German physician Theodor Escherich in 1885 as the 49 “common colon bacillus” (1), Escherichia coli is a Gram-negative facultative 50 anaerobe of the Enterobacteriaceae family and a major component of the normal 51 human intestinal flora (2). Pathogenic E. coli have arisen through the acquisition of 52 large groups of virulence factors on plasmids, prophages and Pathogenicity Islands 53 (PAIs) and are capable of causing both diarrhoeal and extra-intestinal diseases, 54 including Urinary Tract Infections (UTIs), wound infections, meningitis and sepsis (2, 55 3). The Attaching and Effacing (AE) pathogens Enteropathogenic E. coli (EPEC) and 2 56 Enterohaemorrhagic E. coli (EHEC) represent an important subset of pathogenic E. 57 coli and are responsible for significant disease burden worldwide (4). EPEC and 58 EHEC infections are characterised by the formation of ultrastructural lesions on the 59 surface of the gut epithelium known as AE lesions (2, 5). The formation of these 60 lesions, and the infectious process of the AE pathogens more broadly, depends on a 61 Type Three Secretion System (T3SS), encoded by the Locus of Enterocyte 62 Effacement (LEE) PAI (5), and a suite of locally and distally encoded effector proteins 63 (6). The LEE PAI is also found in the AE pathogens Citrobacter rodentium, 64 Escherichia albertii (previously known as Citrobacter freundii and Hafnia alvei 65 respectively) (7, 8) and some Edwardsiella spp. (9, 10). Regulation of the LEE is 66 central to the pathogenesis of AE pathogens, and has been the subject of extensive 67 research. Our review focuses on regulation of the EPEC and EHEC LEE’s, as little is 68 currently known regarding the regulation of the LEE in these other organisms. 69 However, it is likely that many similarities exist between the regulation of these 70 canonical LEE’s and those found in other pathogens. 71 72 73 The Locus of Enterocyte Effacement 74 75 The 35 kb LEE PAI consists of 41 open reading frames (ORFs) and is the defining 76 genetic feature of the AE pathogens. The LEE carries the genes for a T3SS, a 77 number of bacterial effector proteins, chaperones for these effectors, the adhesin 78 intimin and regulatory elements (7). As with many of the virulence-associated 79 elements found in the pathogenic E. coli, the lower G+C content of the LEE (38.3%), 80 in comparison to the E. coli chromosome as a whole, (50.8%) suggests that it was 81 originally acquired by Horizontal Gene Transfer (11, 12). The core LEEs of EPEC 82 and EHEC are 93.9% identical at the nucleotide level, although the EHEC LEE has 3 83 an additional 13 ORFs at the 5’ end due to the insertion of a cryptic prophage (13). 84 However, the sequence of individual genes can be highly variable and ranges from 85 complete invariance between the two organisms in the case of structural components 86 of the T3SS, to 33.52% identity, as is seen for the effector tir (13). 87 88 Organisation of the LEE 89 90 The LEE is arranged as five polycistronic operons: LEE1, LEE2, LEE3, LEE4 and 91 LEE5, a bicistronic operon (grlAB) and multiple monocistronic transcription units (5, 92 14). LEE1 contains the genes encoding the master virulence regulator Ler, the 93 chaperones CesAB, structural components of the T3SS; EscR, EscS, EscT, EscU, 94 EscK (15) and EscL (16). LEE2 encodes for the chaperone CesD, three components 95 of the T3SS machinery, EscC, EscJ and SepD, the effector EspZ and one ORF of 96 unknown function. LEE3 encodes for three components of the T3SS machinery, 97 EscV, EscN and SepQ, the effector EspH and two ORFs of unknown function. LEE4 98 contains the genes encoding the translocator proteins, which form the tip of the T3SS 99 (EspB and EspD) (17), the needle filament EspA (18), the needle protein EscF (19), 100 the chaperone CesD2, the gatekeeper protein SepL (20) and the effector EspF. 101 Finally, LEE5 contains the effectors Tir and intimin, and the chaperone CesT. The 102 effector Map is located 5’ to LEE5 (21) and the effector EspG is located 5’ to LEE1, 103 (22). The addition of the 933L cryptic prophage 3’ to the core LEE genes in EHEC 104 makes the EHEC LEE larger than the EPEC LEE (13). 105 106 Expression of the five LEE operons is controlled through the action of the LEE- 107 encoded transcriptional regulator Ler, the expression of which is regulated through 108 numerous transcription factors in response to an array of intra- and extracellular 109 stimuli, (summarised in Fig.1) which are discussed further below. 110 4 111 112 LEE-encoded regulators: Ler, GrlA and GrlR 113 114 AE pathogens sense and integrate multiple host, environmental and microbiota- 115 derived signals to control expression of the LEE genes, with integration of these 116 diverse signals occurring at the transcriptional, translational and post-translational 117 level (23-28). Below 37°C the global regulator H-NS (29) represses the transcription 118 of the master positive regulator of the LEE, ler (encoded in LEE1) as well as LEE2, 119 LEE3, LEE4 and LEE5 (30-33). However, at 37°C ler is transcribed and, through the 120 coordination of many cues, relieves H-NS mediated repression (30, 34) to allow 121 expression of the LEE genes (23, 35, 36). More than 40 regulators of LEE 122 transcription have been described to date, including both proteins and regulatory 123 RNAs (23, 24, 31, 35, 37-49), not all of which can be discussed here. Many of these 124 systems converge on ler (23, 37) (Fig. 1). 125 126 During Ler-mediated expression, the LEE-encoded regulators GrlA (positive) and 127 GrlR (negative) (38) also influence expression of the LEE. GrlA functions in a similar 128 way to some MerR family transcriptional activators, despite sharing no significant 129 sequence similarity with these proteins (50) and belongs to a novel family of 130 transcriptional activators that also contains a GrlA homologue from Salmonella 131 enterica and CaiF from E. coli and S. flexneri (38, 51). Upon expression, GrlA drives 132 the expression of virulence genes, likely through direct binding to the sub-optimal 18- 133 base-pair spacer between the -10 and -35 elements of the distal LEE1 promoter, P1 134 (Fig. 1B), via a Helix-Turn-Helix (HTH) DNA-binding motif (50, 52). This drives 135 expression of ler and subsequently the entire LEE. Conversely GrlR, cellular levels of 136 which are regulated in a growth phase dependent manner (39), antagonises the 137 system by directly binding to GrlA, preventing its interaction with DNA (53). As 138 demonstrated by Alsharif et al. GrlR is not the only antagonistic mechanism acting 5 139 upon GrlA. GlrA activity is regulated by the sRNA Arl (54) (discussed further below), 140 whilst in some conditions GrlA also requires external queues, such as attachment to 141 host cells and the shear forces which act upon attached bacteria in the gut, to 142 become fully activated and drive ler expression (discussed further below) (26). Thus 143 these auto-regulatory mechanisms allow the LEE to act dynamically by fine-tuning its 144 own expression. 145 146 147 Regulation of the LEE by LysR and Fur-family transcriptional regulators 148 149 LysR-type transcriptional regulators are perhaps the primary family of DNA-binding 150 regulatory proteins in bacteria and interact with DNA through a HTH domain at their 151 amino terminus (55). Two major quorum-sensing responsive LysR-type regulators 152 act upon the LEE, QseA and QseD (Fig. 1B). QseA is part of the LuxS quorum 153 sensing system and responds to microbiota-derived signalling molecules, binding the 154 LEE1 regulatory region, inducing LEE gene expression through increased 155 transcription of ler (40, 56) and direct transcription of LEE1 (57), as well as inducing 156 virulence genes in other areas of the chromosome (58). QseD responds indirectly to 157 Autoinducer-3 (AI-3) and is present across the AE pathogens, but exists almost 158 exclusively in EHEC O157:H7 as a truncated short isoform (sQseD) that lacks the 159 classical HTH domain of the LysR-type regulators (59).