The C-terminal proline-rich repeats of Enteropathogenic E. coli effector EspF are sufficient for the depletion of tight junction membrane proteins and interactions with early and recycling endosomes.

BACKGROUND: Enteropathogenic E. coli (EPEC) causes acute infantile diarrhea accounting for significant morbidity and mortality in developing countries. EPEC uses a type three secretion system to translocate more than twenty effectors into the host intestinal cells. At least four of these effectors, namely EspF, Map, EspG1/G2 and NleA, are reported to disrupt the intestinal tight junction barrier. We have reported earlier that the expression of EspF and Map in MDCK cells causes the depletion of the TJ membrane proteins and compromises the integrity of the intestinal barrier. In the present study, we have examined the role of the proline-rich repeats (PRRs) within the C-terminus of EspF in the depletion of the tight junction membrane proteins and identified key endocytosis markers that interact with EspF via these repeats. RESULTS: We generated mutant EspF proteins which lacked one or more proline-rich repeats (PRRs) from the N-terminus of EspF and examined the effect of their expression on the cellular localization of tight junction membrane proteins. In lysates derived from cells expressing the mutant EspF proteins, we found that the C-terminal PRRs of EspF are sufficient to cause the depletion of TJ membrane proteins. Pull-down assays revealed that the PRRs mediate interactions with the TJ adaptor proteins ZO-1 and ZO-2 as well as with the proteins involved in endocytosis such as caveolin-1, Rab5A and Rab11. CONCLUSIONS: Our study demonstrates the direct role of the proline-rich repeats of EspF in the depletion of the TJ membrane proteins and a possible involvement of the PRRs in the endocytosis of host proteins. New therapeutic strategies can target these PRR domains to prevent intestinal barrier dysfunction in EPEC infections.

Genetic and virulence characteristics of hybrid Shiga toxin-producing and atypical enteropathogenic Escherichia coli strains isolated in South Korea.

Introduction: The predominant hybrid pathogenic E. coli, enterohemorrhagic E. coli (EHEC), combines characteristics of Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC), contributing to global outbreaks with severe symptoms including fatal consequences. Since EHEC infection was designated as a notifiable disease in 2000 in South Korea, around 2000 cases have been reported, averaging approximately 90 cases annually. Aim: In this work, genome-based characteristic analysis and cell-based assay of hybrid STEC/aEPEC strains isolated from livestock feces, animal source foods, and water in South Korea was performed. Methods: To identify the virulence and antimicrobial resistance genes, determining the phylogenetic position of hybrid STEC/aEPEC strains isolated in South Korea, a combination of real-time PCR and whole-genome sequencing (WGS) was used. Additionally, to assess the virulence of the hybrid strains and compare them with genomic characterization, we performed a cell cytotoxicity and invasion assays. Results: The hybrid STEC/aEPEC strains harbored stx and eae genes, encoding Shiga toxins and E. coli attachment/effacement related protein of STEC and EPEC, respectively. Furthermore, all hybrid strains harbored plasmid-carried enterohemolysin(ehxCABD), a key virulence factor in prevalent pathogenic E. coli infections, such as diarrheal disease and hemolytic-uremic syndrome (HUS). Genome-wide phylogenetic analysis revealed a close association between all hybrid strains and specific EPEC strains, suggesting the potential acquisition of Stx phages during STEC/aEPEC hybrid formation. Some hybrid strains showed cytotoxic activity against HeLa cells and invasive properties against epithelial cells. Notably, all STEC/aEPEC hybrids with sequence type (ST) 1,034 (n = 11) exhibited higher invasiveness than those with E2348/69. This highlights the importance of investigating potential correlations between STs and virulence characteristics of E. coli hybrid strains. Conclusion: Through genome-based characterization, we confirmed that the hybrid STEC/aEPEC strains are likely EPEC strains that have acquired STEC virulence genes via phage. Furthermore, our results emphasize the potential increased danger to humans posed by hybrid STEC/aEPEC strains isolated in South Korea, containing both stx and eaeA, compared to STEC or EPEC alone.

A dual-channel electrochemical biosensor enables concurrent detection of pathogens and antibiotic resistance.

Diarrheagenic E. coli infections, commonly treated with ß-lactam antibiotics, contribute to antibiotic resistance - a pressing public health concern. Rapid monitoring of pathogen antibiotic resistance is vital to combat antimicrobial spread. Current bacterial diagnosis methods identify pathogens or determine antibiotic resistance separately, necessitating multiple assays. There is an urgent need for tools that simultaneously identify infectious agents and their antibiotic resistance at the point of care (POC). We developed an integrated electrochemical chip-based biosensor for detecting enteropathogenic E. coli (EPEC), a major neonatal diarrheal pathogen, using an antibody against a virulence marker, termed EspB, and the ß-lactam resistance marker, ß-lactamase. A dual-channel microfabricated chip, bio-functionalized with a specific EspB monoclonal antibody, and nitrocefin, a ß -lactamase substrate was utilized. The chip facilitated electrochemical impedance spectroscopy (EIS)-based detection of EspB antigen and EspB-expressing bacteria. For ß-lactam resistance profiling, a second channel enabled differential-pulse voltammetric (DPV) measurement of hydrolyzed nitrocefin. EIS-based detection of EspB antigen was calibrated (LOD: 4.3 ng/mL ±1 and LOQ: 13.0 ng/mL ±3) as well as DPV-based detection of the antibiotic resistance marker, ß-lactamase (LOD: 3.6 ng/mL ±1.65 and LOQ: 10 ng/mL ±4). The integrated EIS and DPV biosensor was employed for the simultaneous detection of EspB-expressing and ß-lactamase-producing bacteria. The combined readout from both channels allowed the distinction between antibiotic-resistant and -sensitive pathogenic bacteria. The integrated electrochemical biosensor successfully achieved simultaneous, rapid detection of double positive EspB- and ß-lactamase-expressing bacteria. Such distinction enabled by a portable device within a short assay time and a simplified sample preparation, may be highly valuable in mitigating the spread of AMR. This new diagnostic tool holds promise for the development of POC devices in clinical diagnosis.

O26 Polysaccharides as Key Players in Enteropathogenic E. coli Immune Evasion and Vaccine Development.

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

O26 Polysaccharides as key players in enteropathogenic E. coli immune evasion and vaccine development

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

Enteropathogenic E. coli infection co-elicits lysosomal exocytosis and lytic host cell death.

IMPORTANCE: Enteropathogenic Escherichia coli (EPEC) infection is a significant cause of gastroenteritis, mainly in children. Therefore, studying the mechanisms of EPEC infection is an important research theme. EPEC modulates its host cell life by injecting via a type III secretion machinery cell death modulating effector proteins. For instance, while EspF and Map promote mitochondrial cell death, EspZ antagonizes cell death. We show that these effectors also control lysosomal exocytosis, i.e., the trafficking of lysosomes to the host cell plasma membrane. Interestingly, the capacity of these effectors to induce or protect against cell death correlates completely with their ability to induce LE, suggesting that the two processes are interconnected. Modulating host cell death is critical for establishing bacterial attachment to the host and subsequent dissemination. Therefore, exploring the modes of LE involvement in host cell death is crucial for elucidating the mechanisms underlying EPEC infection and disease.

Enteropathogenic Escherichia coli is a predominant pathotype in healthy pigs in Hubei Province of China.

AIM: This study aims to investigate the prevalence of intestinal pathogenic Escherichia coli (InPEC) in healthy pig-related samples and evaluate the potential virulence of the InPEC strains. METHODS AND RESULTS: A multiplex PCR method was established to identify different pathotypes of InPEC. A total of 800 rectal swab samples and 296 pork samples were collected from pig farms and slaughterhouses in Hubei province, China. From these samples, a total of 21 InPEC strains were isolated, including 19 enteropathogenic E. coli (EPEC) and 2 shiga toxin-producing E. coli (STEC) strains. By whole-genome sequencing and in silico typing, it was shown that the sequence types and serotypes were diverse among the strains. Antimicrobial susceptibility assays showed that 90.48% of the strains were multi-drug resistant. The virulence of the strains was first evaluated using the Galleria mellonella larvae model, which showed that most of the strains possessed medium to high pathogenicity. A moderately virulent EPEC isolate was further selected to characterize its pathogenicity using a mouse model, which suggested that it could cause significant diarrhea. Bioluminescence imaging (BLI) was then used to investigate the colonization dynamics of this EPEC isolate, which showed that the EPEC strain could colonize the mouse cecum for up to 5 days.

Molecular and Genomic Analysis of the Virulence Factors and Potential Transmission of Hybrid Enteropathogenic and Enterotoxigenic Escherichia coli (EPEC/ETEC) Strains Isolated in South Korea.

Hybrid strains Escherichia coli acquires genetic characteristics from multiple pathotypes and is speculated to be more virulent; however, understanding their pathogenicity is elusive. Here, we performed genome-based characterization of the hybrid of enteropathogenic (EPEC) and enterotoxigenic E. coli (ETEC), the strains that cause diarrhea and mortality in children. The virulence genes in the strains isolated from different sources in the South Korea were identified, and their phylogenetic positions were analyzed. The EPEC/ETEC hybrid strains harbored eae and est encoding E. coli attaching and effacing lesions and heat-stable enterotoxins of EPEC and ETEC, respectively. Genome-wide phylogeny revealed that all hybrids (n = 6) were closely related to EPEC strains, implying the potential acquisition of ETEC virulence genes during ETEC/EPEC hybrid emergence. The hybrids represented diverse serotypes (O153:H19 (n = 3), O49:H10 (n = 2), and O71:H19 (n = 1)) and sequence types (ST546, n = 4; ST785, n = 2). Furthermore, heat-stable toxin-encoding plasmids possessing estA and various other virulence genes and transporters, including nleH2, hlyA, hlyB, hlyC, hlyD, espC, espP, phage endopeptidase Rz, and phage holin, were identified. These findings provide insights into understanding the pathogenicity of EPEC/ETEC hybrid strains and may aid in comparative studies, virulence characterization, and understanding evolutionary biology.

Topology and function of translocated EspZ.

EspZ and Tir are essential virulence effectors of enteropathogenic Escherichia coli (EPEC). EspZ, the second translocated effector, has been suggested to antagonize host cell death induced by the first translocated effector, Tir (translocated intimin receptor). Another characteristic of EspZ is its localization to host mitochondria. However, studies that explored the mitochondrial localization of EspZ have examined the ectopically expressed effector and not the more physiologically relevant translocated effector. Here, we confirmed the membrane topology of translocated EspZ at infection sites and the involvement of Tir in confining its localization to these sites. Unlike the ectopically expressed EspZ, the translocated EspZ did not colocalize with mitochondrial markers. Moreover, no correlation has been found between the capacity of ectopically expressed EspZ to target mitochondria and the ability of translocated EspZ to protect against cell death. Translocated EspZ may have to some extent diminished F-actin pedestal formation induced by Tir but has a marked effect on protecting against host cell death and on promoting host colonization by the bacteria. Taken together, our results suggest that EspZ plays an essential role in facilitating bacterial colonization, likely by antagonizing cell death mediated by Tir at the onset of bacterial infection. This activity of EspZ, which occurs by targeting host membrane components at infection sites, and not mitochondria, may contribute to successful bacterial colonization of the infected intestine. IMPORTANCE EPEC is an important human pathogen that causes acute infantile diarrhea. EspZ is an essential virulence effector protein translocated from the bacterium into the host cells. Detailed knowledge of its mechanisms of action is, therefore, critical for better understanding the EPEC disease. We show that Tir, the first translocated effector, confines the localization of EspZ, the second translocated effector, to infection sites. This activity is important for antagonizing the pro-cell death activity conferred by Tir. Moreover, we show that translocated EspZ leads to effective bacterial colonization of the host. Hence, our data suggest that translocated EspZ is essential because it confers host cell survival to allow bacterial colonization at an early stage of bacterial infection. It performs these activities by targeting host membrane components at infection sites. Identifying these targets is critical for elucidating the molecular mechanism underlying the EspZ activity and the EPEC disease.

Atypical enteropathogenic E. coli are associated with disease activity in ulcerative colitis.

With increasing urbanization and industrialization, the prevalence of inflammatory bowel diseases (IBDs) has steadily been rising over the past two decades. IBD involves flares of gastrointestinal (GI) inflammation accompanied by microbiota perturbations. However, microbial mechanisms that trigger such flares remain elusive. Here, we analyzed the association of the emerging pathogen atypical enteropathogenic E. coli (aEPEC) with IBD disease activity. The presence of diarrheagenic E. coli was assessed in stool samples from 630 IBD patients and 234 age- and sex-matched controls without GI symptoms. Microbiota was analyzed with 16S ribosomal RNA gene amplicon sequencing, and 57 clinical aEPEC isolates were subjected to whole-genome sequencing and in vitro pathogenicity experiments including biofilm formation, epithelial barrier function and the ability to induce pro-inflammatory signaling. The presence of aEPEC correlated with laboratory, clinical and endoscopic disease activity in ulcerative colitis (UC), as well as microbiota dysbiosis. In vitro, aEPEC strains induce epithelial p21-activated kinases, disrupt the epithelial barrier and display potent biofilm formation. The effector proteins espV and espG2 distinguish aEPEC cultured from UC and Crohn's disease patients, respectively. EspV-positive aEPEC harbor more virulence factors and have a higher pro-inflammatory potential, which is counteracted by 5-ASA. aEPEC may tip a fragile immune-microbiota homeostasis and thereby contribute to flares in UC. aEPEC isolates from UC patients display properties to disrupt the epithelial barrier and to induce pro-inflammatory signaling in vitro.

EspH interacts with the host active Bcr related (ABR) protein to suppress RhoGTPases.

Enteropathogenic Escherichia coli are bacterial pathogens that colonize the gut and cause severe diarrhea in humans. Upon intimate attachment to the intestinal epithelium, these pathogens translocate via a type III secretion system virulent proteins, termed effectors, into the host cells. These effectors manipulate diverse host cell organelles and functions for the pathogen's benefit. However, the precise mechanisms underlying their activities are not fully understood despite intensive research. EspH, a critical effector protein, has been previously reported to disrupt the host cell actin cytoskeleton by suppressing RhoGTPase guanine exchange factors. However, native host proteins targeted by EspH to mediate these activities remained unknown. Here, we identified the active Bcr related (ABR), a protein previously characterized to possess dual Rho guanine nucleotide exchange factor and GTPase activating protein (GAP) domains, as a native EspH interacting partner. These interactions are mediated by the effector protein's C-terminal 38 amino acid segment. The effector primarily targets the GAP domain of ABR to suppress Rac1 and Cdc42, host cell cytotoxicity, bacterial invasion, and filopodium formation at infection sites. Knockdown of ABR expression abolished the ability of EspH to suppress Rac1, Cdc42. Our studies unravel a novel mechanism by which host RhoGTPases are hijacked by bacterial effectors.

Enteropathogenic Escherichia coli (EPEC) expressing a non-functional bundle-forming pili (BFP) also leads to increased growth failure and intestinal inflammation in C57BL/6 mice.

Bundle-forming pili (BFP) are implicated in the virulence of typical enteropathogenic E. coli (EPEC), resulting in enhanced colonization and mild to severe disease outcomes; hence, non-functional BFP may have a major influence on disease outcomes in vivo. Weaned antibiotic pre-treated C57BL/6 mice were orally infected with EPEC strain UMD901 (E2348/69 bfpA C129S); mice were monitored daily for body weight; stool specimens were collected daily; and intestinal tissues were collected at the termination of the experiment on day 3 post-infection. Real-time PCR was used to quantify fecal shedding and tissue burden. Intestinal inflammatory biomarkers lipocalin-2 (LCN-2) and myeloperoxidase (MPO) were also assessed. Infection caused substantial body weight loss, bloody diarrhea, and intestinal colonization with fecal and intestinal tissue inflammatory biomarkers that were comparable to those previously published with the wild-type typical EPEC strain. Here we further report on the evaluation of an EPEC infection model, showing how disruption of bfp function does not impair, and may even worsen diarrhea, colonization, and intestinal disruption and inflammation. More research is needed to understand the role of bfp in pathogenicity of EPEC infections in vivo.

Activation of the Type III Secretion System of Enteropathogenic Escherichia coli Leads to Remodeling of Its Membrane Composition and Function.

The cell envelope of Gram-negative bacteria is a complex structure, essential for bacterial survival and for resistance to many antibiotics. Channels that cross the bacterial envelope and the host cell membrane form secretion systems that are activated upon attachment to host, enabling bacteria to inject effector molecules into the host cell, required for bacterium-host interaction. The type III secretion system (T3SS) is critical for the virulence of several pathogenic bacteria, including enteropathogenic Escherichia coli (EPEC). EPEC T3SS activation is associated with repression of carbon storage regulator (CsrA), resulting in gene expression remodeling, which is known to affect EPEC central carbon metabolism and contributes to the adaptation to a cell-adherent lifestyle in a poorly understood manner. We reasoned that the changes in the bacterial envelope upon attachment to the host and the activation of a secretion system may involve a modification of the lipid composition of bacterial envelope. Accordingly, we performed a lipidomics analysis on mutant strains that simulate T3SS activation. We saw a shift in glycerophospholipid metabolism toward the formation of lysophospholipids, attributed to corresponding upregulation of the phospholipase gene pldA and the acyltransferase gene ygiH upon T3SS activation in EPEC. We also detected a shift from menaquinones and ubiquinones to undecaprenyl lipids, concomitant with abnormal synthesis of O antigen. The remodeling of lipid metabolism is mediated by CsrA and associated with increased bacterial cell size and zeta potential and a corresponding alteration in EPEC permeability to vancomycin, increasing the sensitivity of T3SS-activated strains and of adherent wild-type EPEC to the antibiotic. IMPORTANCE The characterization of EPEC membrane lipid metabolism upon attachment to the host is an important step toward a better understanding the shift of EPEC, a notable human pathogen, from a planktonic to adherent lifestyle. It may also apply to other pathogenic bacteria that use this secretion system. We predict that upon attachment to host cells, the lipid remodeling upon T3SS activation contributes to bacterial fitness and promotes host colonization, and we show that it is associated with increased cell permeability and higher sensitivity to vancomycin. To the best of our knowledge, this is the first demonstration of a bacterial lipid remodeling due to activation of a secretion system.

Genetic and phylogenetic characterization of Shiga toxin-producing Escherichia coli and enteropathogenic E. coli from livestock in Jiangsu by using whole-genome sequencing.

AIMS: There are knowledge gaps regarding STEC and EPEC strains in livestock in Jiangsu, China. This study aimed to evaluate the potential public health significance of STEC and EPEC strains isolated from livestock by determining the serotypes, virulence profiles, and genetic relationship with international STEC strains. METHODS AND RESULTS: A total of 68 STEC and 37 EPEC strains were obtained from 231 faecal sheep samples and 70 faecal cattle samples. By using whole-genome sequencing (WGS) analysis, all STEC belonged to 15 O: H serotypes, and the most prevalent serotypes were O6:H10 (19.1%), O155:H21 (14.7%), and O21:H25 (10.3%). The main Shiga toxin gene subtypes detected were stx1c (41.2%), stx1a (26.5%), stx2b (14.7%), and stx2k (14.7%). Only the STEC from cattle carried eae gene. Other adherence-associated or toxin-related genes, including lpfA (70.6%), iha (48.5%), subA (54.4%), and ehxA (33.8%), were found in STEC. All EPEC strains were bfpA-negative, and the predominant eae variants were eae-ß1 (62.2%), eae-ζ (21.6%), and eae-θ (8.1%). The core-genome multi-locus sequence typing (cgMLST) analysis revealed nine scattered clusters in STEC and one dominant cluster in EPEC. The strains with the same serotypes, including O22:H8 and O43:H2 in the two towns, possessed a closely genomic distance. The core genome single-nucleotide polymorphism (cgSNP) showed that part of STEC strains in this study was clustered with isolates possessing the same serotypes from the Netherlands, Sweden, and Xinjiang of China. Five serotypes of STEC isolates were associated with the clinical STEC strains from databases. CONCLUSION: This study provided the diverse serotypes and the virulence genes profiles in STEC and EPEC strains. Local strains possessed widely diverse and scattered clusters by cgMLST. Closely genomic correlation with clinical isolates displayed that part of the STEC strains may threaten to public health. SIGNIFICANCE AND IMPACT OF THE STUDY: Non-O157 STEC strains act as important pathogens for human infections. This study supports the increased surveillance work of non-O157 STEC rather than just O157 STEC in this region.

NEDD8 Deamidation Inhibits Cullin RING Ligase Dynamics.

Cullin-RING ligases (CRLs) are a significant subset of Ubiquitin E3 ligases that regulate multiple cellular substrates involved in innate immunity, cytoskeleton modeling, and cell cycle. The glutamine deamidase Cycle inhibitory factor (Cif) from enteric bacteria inactivates CRLs to modulate these processes in the host cell. The covalent attachment of a Ubiquitin-like protein NEDD8 catalytically activates CRLs by driving conformational changes in the Cullin C-terminal domain (CTD). NEDDylation results in a shift from a compact to an open CTD conformation through non-covalent interactions between NEDD8 and the WHB subdomain of CTD, eliminating the latter's inhibitory interactions with the RING E3 ligase-Rbx1/2. It is unknown whether the non-covalent interactions are sufficient to stabilize Cullin CTD's catalytic conformation. We studied the dynamics of Cullin-CTD in the presence and absence of NEDD8 using atomistic molecular dynamics (MD) simulations. We uncovered that NEDD8 engages in non-covalent interactions with 4HB/αß subdomains in Cullin-CTD to promote open conformations. Cif deamidates glutamine 40 in NEDD8 to inhibit the conformational change in CRLs by an unknown mechanism. We investigated the effect of glutamine deamidation on NEDD8 and its interaction with the WHB subdomain post-NEDDylation using MD simulations and NMR spectroscopy. Our results suggest that deamidation creates a new intramolecular salt bridge in NEDD8 to destabilize the NEDD8/WHB complex and reduce CRL activity.

Biosynthesis of UDP-2-acetamido-4-formamido-2,4,6-trideoxy-hexose by WekG, WekE, WekF, and WekD: Enzymes in the Wek pathway responsible for O-antigen Assembly in Escherichia coli O119.

Considering the importance of bacterial glycoconjugates on virulence and host mimicry, there is a need to better understand the biosynthetic pathways of these unusual sugars to identify critical targets involved in bacterial pathogenesis. In this report, we describe the cloning, overexpression, purification, and biochemical characterization of the four central enzymes in the biosynthesis pathway for UDP-2-acetamido-4-formamido-2,4,6-trideoxy-hexose, WekG, WekE, WekF, and WekD. Product peaks from enzyme-substrate reactions were detected by using a combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). Putative enzyme assignments were provided by protein sequence analysis. Combined with the mass spectrometric characterization of pathway intermediates, we propose a biosynthetic pathway for UDP-2-acetamido-4-formamido-2,4,6-trideoxy-hexose. This process involves C-4, C-6 dehydration, C-4 amination, and formylation. CID-ESI-MSn result confirmed that the final product is a 4 formamido derivative too rather than the 3 formamido derivatives as reported earlier.

Role of the SOS Response in the Generation of Antibiotic Resistance In Vivo.

The SOS response to DNA damage is a conserved stress response in Gram-negative and Gram-positive bacteria. Although this pathway has been studied for years, its relevance is still not familiar to many working in the fields of clinical antibiotic resistance and stewardship. Under some conditions, the SOS response favors DNA repair and preserves the genetic integrity of the organism. On the other hand, the SOS response also includes induction of error-prone DNA polymerases, which can increase the rate of mutation, called the mutator phenotype or "hypermutation." As a result, mutations can occur in genes conferring antibiotic resistance, increasing the acquisition of resistance to antibiotics. Almost all of the work on the SOS response has been on bacteria exposed to stressors in vitro. In this study, we sought to quantitate the effects of SOS-inducing drugs in vivo, in comparison with the same drugs in vitro. We used a rabbit model of intestinal infection with enteropathogenic Escherichia coli strain E22. SOS-inducing drugs triggered the mutator phenotype response in vivo as well as in vitro. Exposure of E. coli strain E22 to ciprofloxacin or zidovudine, both of which induce the SOS response in vitro, resulted in increased antibiotic resistance to 3 antibiotics: rifampin, minocycline, and fosfomycin. Zinc was able to inhibit the SOS-induced emergence of antibiotic resistance in vivo, as previously observed in vitro. Our findings may have relevance in reducing the emergence of resistance to new antimicrobial drugs.

Development and validation of a high-content screening assay for inhibitors of enteropathogenic E. coli adhesion.

Enteropathogenic E. coli (EPEC) causes intestinal infections leading to severe diarrhea. EPEC attaches to the host cell causing lesions to the intestinal epithelium coupled with the effacement of microvilli. In the process, actin accumulates into a pedestal-like structure under bacterial microcolonies. We designed an automated fluorescence microscopy-based screening method for discovering compounds capable of inhibiting EPEC adhesion and virulence using aurodox, a type three secretion system (T3SS) inhibitor, as a positive control. The screening assay employs an EPEC strain (2348/69) expressing a fluorescent protein and actin staining for monitoring the bacteria and their pedestals respectively, analyzing these with a custom image analysis pipeline. The assay allows for the discovery of compounds capable of preventing the formation of pathogenic actin rearrangements. These compounds may be interfering with virulence-related molecular pathways relevant for developing antivirulence leads.

Lambda Red-Mediated Recombination in Shiga Toxin-Producing Escherichia coli.

The bacteriophage Lambda (λ) "Red" recombination system has enabled the development of efficient methods for engineering bacterial chromosomes. This system has been particularly important to the field of bacterial pathogenesis, where it has advanced the study of virulence factors from Shiga toxin-producing and enteropathogenic Escherichia coli (STEC and EPEC). Transient plasmid-driven expression of Lambda Red allows homologous recombination between PCR-derived linear DNA substrates and target loci in the STEC/EPEC chromosomes. Red-associated techniques can be used to create individual gene knockouts, generate deletions of large pathogenicity islands, and make markerless allelic exchanges. This chapter describes specific strategies and procedures for performing Lambda Red-mediated genome engineering in STEC.

Efficacy of novel phages for control of multi-drug resistant Escherichia coli O177 on artificially contaminated beef and their potential to disrupt biofilm formation.

Contaminated beef is a prominent source of foodborne pathogens such as Escherichia coli O177. Susceptibility of nine multi-drug resistant E. coli O177 strains against eight individual phages and six phage cocktails was assessed using polystyrene microplate titer plate. Further, 180 beef samples were independently inoculated with E. coli O177 cells in triplicates and treated with eight individual phages and six phage cocktails to determine their efficacy in inhibiting bacteria growth at 4 °C over a 7-day incubation period. Results revealed that all E. coli O177 strains were susceptible to the phages. A significant log reduction in viable E. coli O177 cell counts was observed on beef samples upon phage treatment over the 7-day incubation period. Two individual phages and three phage cocktails reduced E. coli cell counts to levels below the detection limit (1.0 log10 CFU/g). Log reduction of viable E. coli cell counts ranged from 2.10 to 7.81 CFU/g for individual phages and from 2.86 to 7.81 CFU/g for cocktails. Individual phages and phage cocktails inhibited E. coli O177 biofilm formation with phage cocktails showing high efficacy. Furthermore, phage cocktails showed greater efficacy in destroying pre-formed biofilm than individual phages. Based on these findings, we concluded that phage cocktails developed in this study could be used to reduce E. coli O177 contamination and extend the shelf-life of stored raw beef.