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Intestinal parasites are part of the intestinal ecosystem and have been shown to establish close interactions with the intestinal microbiota. However, little is known about the influence of intestinal protozoa on the regulation of the immune response. In this study, we analyzed the regulation of the immune response of germ-free mice transplanted with fecal microbiota (FMT) from individuals with multiple parasitic protozoans (P) and non-parasitized individuals (NP). We determined the production of intestinal cytokines, the lymphocyte populations in both the colon and the spleen, and the genetic expression of markers of intestinal epithelial integrity. We observed a general downregulation of the intestinal immune response in mice receiving FMT-P. We found significantly lower intestinal production of the cytokines IL-6, TNF, IFN-γ, MCP-1, IL-10, and IL-12 in the FMT-P. Furthermore, a significant decrease in the proportion of CD3+, CD4+, and Foxp3+ T regulatory cells (Treg) was observed in both, the colon and spleen with FMT-P in contrast to FMT-NP. We also found that in FMT-P mice there was a significant decrease in tjp1 expression in all three regions of the small intestine; ocln in the ileum; reg3γ in the duodenum and relmβ in both the duodenum and ileum. We also found an increase in colonic mucus layer thickness in mice colonized with FMT-P in contrast with FMT-NP. Finally, our results suggest that gut protozoa, such as Blastocystis hominis, Entamoeba coli, Endolimax nana, Entamoeba histolytica/E. dispar, Iodamoeba bütschlii, and Chilomastix mesnili consortia affect the immunoinflammatory state and induce functional changes in the intestine via the gut microbiota. Likewise, it allows us to establish an FMT model in germ-free mice as a viable alternative to explore the effects that exposure to intestinal parasites could have on the immune response in humans., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Partida-Rodríguez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Prospective data from sub-Saharan Africa suggests that treatment outcomes for people living with HIV (PWH) with Hodgkin lymphoma (HL) are similar to those without HIV. However, real-world data from high-resource settings and retrospective studies from sub-Saharan Africa, suggest inferior outcomes. We set out to evaluate the real-world treatment outcomes for HL in South Africa to better understand the disparate outcomes., Methods: We established a prospective, observational cohort of newly diagnosed, adult (≥ 18 years) HL cases recruited from Chris Hani Baragwanath Academic and Netcare Olivedale Hospitals in Johannesburg, South Africa between March 2021 and March 2023. Participants were followed for up to 18 months after enrollment with data censored on December 23rd, 2023. The primary endpoint was 1-year overall survival., Results: We enrolled 47 participants with HL including 31 PWH and 16 HIV-negative. Advanced stage disease and B symptoms were common at time of diagnosis irrespective of HIV status. Bone marrow biopsy, performed during the work-up and evaluation of cytopenias, provided the initial diagnosis of HL in 16/31 (52%) PWH. HIV status and bone marrow involvement were associated with early mortality (within 3 months of diagnosis) and a poorer 1-year overall survival from diagnosis (HIV: 55% vs. 88%; p = 0.03; bone marrow involvement: 50% vs. 80%; p = 0.02). Among evaluable participants, those that received at least 6 cycles of chemotherapy and underwent response assessment, there was no difference between those with and without HIV., Conclusion: Traditional laboratory markers of poor prognosis including anemia, lymphopenia and hypoalbuminemia were more common among PWH and those with bone marrow involvement and suggest high risk disease. A better understanding of the drivers of these aggressive presentations is warranted to ensure more PWH are able to tolerate chemotherapy., (© 2024. The Author(s).)

Enterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC's ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC's response to metabolites from an in vitro culture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficile infection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP , spy , soxS , yhcN , and bhsA ) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.

The gastrointestinal (GI) environment plays a critical role in shaping enteric infections. Host environmental factors create bottlenecks, restrictive events that reduce the genetic diversity of invading bacterial populations. However, the identity and impact of bottleneck events on bacterial infection are largely unknown. We used Citrobacter rodentium infection of mice, a model of human pathogenic Escherichia coli infections, to examine bacterial population dynamics and quantify bottlenecks to host colonization. Using Sequence Tag-based Analysis of Microbial Populations (STAMP) we characterized the founding population size (Nb') and relatedness of C. rodentium populations at relevant tissue sites during early- and peak-infection. We demonstrate that the GI environment severely restricts the colonizing population, with an average Nb' of only 12-43 lineages (of 2,000+ inoculated) identified regardless of time or biogeographic location. Passage through gastric acid and escape to the systemic circulation were identified as major bottlenecks during C. rodentium colonization. Manipulating such events by increasing gastric pH dramatically increased intestinal Nb'. Importantly, removal of the stomach acid barrier had downstream consequences on host systemic colonization, morbidity, and mortality. These findings highlight the capability of the host GI environment to limit early pathogen colonization, controlling the population of initial founders with consequences for downstream infection outcomes., (© 2022. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Objective: Fine needle aspiration (FNA) is an early step in the work-up of lymphadenopathy in people with HIV (PWH). We set out to characterize the FNA cytology in PWH and report on the time to lymphoma diagnosis through the FNA clinics in the public healthcare system in Johannesburg, South Africa., Design: Retrospective review of laboratory database., Methods: A retrospective chart review of patients undergoing FNA through the department of cytopathology at the National Health Laboratory Service (NHLS) was undertaken. Results of FNAs performed between March and May 2018 were reviewed. Medical record chart abstraction included general demographics, HIV status, site and results of FNA, prior history of malignancy and other laboratory data., Results: Five hundred and thirty-nine lymph node FNAs were performed on PWH. Pathological findings included tuberculosis 47% (252), inadequate sampling 14% (75), reactive adenopathy 13% (71), benign disease 12% (63), suspicious for lymphoproliferative neoplasm 8% (45), other malignancy 4% (21) and inflammation 2% ( n  = 12). Only 53% (24) of lymphomas were confirmed by biopsy. Those not confirmed had a high mortality (57%) and loss to follow-up rate (29%) over the following year. The median diagnostic interval exceeded 8 weeks from time of FNA to lymphoma diagnosis., Conclusion: FNA is an important screening modality in this high HIV and tuberculosis (TB) burden region. Patients with cytology suggestive for lymphoma, but without biopsy confirmation, have a high mortality rate suggesting undiagnosed lymphoma. A better understanding of the barriers to appropriate diagnostic triage for lymphoma is needed., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

The type VI secretion system (T6SS) is a contractile nanomachine widely distributed among pathogenic and commensal Gram-negative bacteria. The T6SS is used for inter-bacterial competition to directly kill competing species; however, its importance during bacterial infection in vivo remains poorly understood. We report that the murine pathogen Citrobacter rodentium, used as a model for human pathogenic Escherichia coli, harbors two functional T6SSs. C. rodentium employs its T6SS-1 to colonize the murine gastrointestinal tract by targeting commensal Enterobacteriaceae. We identify VgrG1 as a C. rodentium T6SS antibacterial effector, which exhibits toxicity in E. coli. Conversely, commensal prey species E. coli Mt1B1 employs two T6SSs of its own to counter C. rodentium colonization. Collectively, these data demonstrate that the T6SS is a potent weapon during bacterial competition and is used by both invading pathogens and resident microbiota to fight for a niche in the hostile gut environment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Child undernutrition is a global health issue associated with a high burden of infectious disease. Undernourished children display an overabundance of intestinal pathogens and pathobionts, and these bacteria induce enteric dysfunction in undernourished mice; however, the cause of their overgrowth remains poorly defined. Here, we show that disease-inducing human isolates of Enterobacteriaceae and Bacteroidales spp. are capable of multi-species symbiotic cross-feeding, resulting in synergistic growth of a mixed community in vitro. Growth synergy occurs uniquely under malnourished conditions limited in protein and iron: in this context, Bacteroidales spp. liberate diet- and mucin-derived sugars and Enterobacteriaceae spp. enhance the bioavailability of iron. Analysis of human microbiota datasets reveals that Bacteroidaceae and Enterobacteriaceae are strongly correlated in undernourished children, but not in adequately nourished children, consistent with a diet-dependent growth synergy in the human gut. Together these data suggest that dietary cross-feeding fuels the overgrowth of pathobionts in undernutrition., (© 2021. The Author(s).)

Purpose: Diagnosis of AIDS lymphoma in low-resource settings, like South Africa, is often delayed, leaving patients with limited treatment options. In tuberculosis (TB) endemic regions, overlapping signs and symptoms often lead to diagnostic delays. Assessment of plasma cell-free DNA (cfDNA) by next-generation sequencing (NGS) may expedite the diagnosis of lymphoma but requires high-quality cfDNA., Methods: People living with HIV with newly diagnosed aggressive B-cell lymphoma and those with newly diagnosed TB seeking care at Chris Hani Baragwanath Academic Hospital and its surrounding clinics, in Soweto, South Africa, were enrolled in this study. Each participant provided a whole blood specimen collected in cell-stabilizing tubes. Quantity and quality of plasma cfDNA were assessed. NGS of the immunoglobulin heavy chain was performed., Results: Nine HIV+ patients with untreated lymphoma and eight HIV+ patients with TB, but without lymphoma, were enrolled. All cfDNA quantity and quality metrics were similar between the two groups, except that cfDNA accounted for a larger fraction of recovered plasma DNA in patients with lymphoma. The concentration of cfDNA in plasma also trended higher in patients with lymphoma. NGS of immunoglobulin heavy chain showed robust amplification of DNA, with large amplicons (> 250 bp) being more readily detected in patients with lymphoma. Clonal sequences were detected in five of nine patients with lymphoma, and none of the patients with TB., Conclusion: This proof-of-principle study demonstrates that whole blood collected for cfDNA in a low-resource setting is suitable for sophisticated sequencing analyses, including clonal immunoglobulin NGS. The detection of clonal sequences in more than half of patients with lymphoma shows promise as a diagnostic marker that may be explored in future studies., Competing Interests: Moosa PatelHonoraria: Roche/Genentech, Novartis, Janssen, Novartis South Africa, Amgen, RocheConsulting or Advisory Role: Janssen Research & Development, Pfizer, Janssen, NovartisResearch Funding: RocheTravel, Accommodations, Expenses: Janssen, Roche, Novartis Vinitha PhilipTravel, Accommodations, Expenses: Roche Philippa AshmoreConsulting or Advisory Role: Novartis, Janssen, TakedaTravel, Accommodations, Expenses: Takeda, Key Oncologics, Cipla, Sanofi Wendy StevensResearch Funding: Roche, Abbott Laboratories, Cepheid Nina Wagner-JohnstonConsulting or Advisory Role: ADC Therapeutics, Bayer, Regeneron, Calibr, Verastem, Gilead SciencesResearch Funding: Merck, Novartis/Pfizer, Genentech, Astex Pharmaceuticals, Juno Therapeutics, Regeneron, Acerta Pharma, ADC Therapeutics Christopher D. GockeLeadership: OncoMEDx IncStock and Other Ownership Interests: OncoMEDx IncPatents, Royalties, Other Intellectual Property: Intellectual property licensed from Penn State to my company, OncoMEDx, Inc Neil A. MartinsonResearch Funding: Pfizer Rena R. XianHonoraria: InvivoscribeTravel, Accommodations, Expenses: InvivoscribeNo other potential conflicts of interest were reported.

Purpose: The aim of this study was to review the current status of clinical trials for HIV-associated malignancies in people living with HIV in sub-Saharan Africa (SSA) and efforts made by the AIDS Malignancy Consortium (AMC) to build capacity in SSA for HIV malignancy research., Methods: All malignancy-related clinical trials in 49 SSA countries on ClinicalTrials.gov were reviewed and evaluated for inclusion and exclusion criteria pertaining to HIV status. Additional studies by AMC in SSA were compiled from Web-based resources, and narrative summaries were prepared to highlight AMC capacity building and training initiatives., Results: Of 96 cancer trials identified in SSA, only 11 focused specifically on people living with HIV, including studies in Kaposi sarcoma, cervical dysplasia and cancer, non-Hodgkin lymphoma, and ocular surface squamous neoplasia. Recognizing the increasing cancer burden in the region, AMC expanded its clinical trial activities to SSA in 2010, with 4 trials completed to date and 6 others in progress or development, and has made ongoing investments in developing research infrastructure in the region., Conclusion: As the HIV-associated malignancy burden in SSA evolves, research into this domain has been limited. AMC, the only global HIV malignancy-focused research consortium, not only conducts vital HIV-associated malignancies research in SSA, but also develops pathology, personnel, and community-based infrastructure to meet these challenges in SSA. Nonetheless, there is an ongoing need to build on these efforts to improve HIV-associated malignancies outcomes in SSA., Competing Interests: The opinions expressed in this article are the authors own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States Government. The other authors report no proprietary or commercial conflicts of interest with respect to any product mentioned or concept discussed in the present work.

Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx-regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens., (© 2018 John Wiley & Sons Ltd.)

The redox balance in a variety of Gram-negative bacteria was explored using redox sensitive GFP (roGFP2), J. van der Heijden et al. doi:10.1016/j.freeradbiomed.2015.11.029[1]. This data article provides Supporting material to further investigate the relationship between Salmonella typhimurium survival and oxidative stress. The first set of data presented in this article, shows the percentage of surviving bacteria after exposure to hydrogen peroxide. The second set of data shows the concentration of hydrogen peroxide that was produced by S. Typhimurium in different growth phases. The last set of data shows the percentage of surviving S. Typhimurium bacteria after exposure to different antibiotics.

The Escherichia coli genome encodes approximately 30 two-component systems that are required for sensing and responding to a variety of environmental and physiological cues. Recent studies have revealed numerous regulatory connections between two-component systems and small noncoding RNAs (sRNAs), which posttranscriptionally regulate gene expression by base pairing with target mRNAs. In this study, we investigated the role of sRNAs in the CpxAR two-component system, which detects and mediates an adaptive response to potentially lethal protein misfolding in the Gram-negative bacterial envelope. Here, we showed for the first time that sRNAs are members of the Cpx regulon. We found that CpxR binds to the promoter regions and regulates expression of two sRNA genes, cyaR and rprA. We also investigated the roles that these sRNAs play in the Cpx response. Cpx repression of cyaR expression creates a feed-forward loop, in which CpxAR increases expression of the inner membrane protein YqaE both directly at the transcriptional level and indirectly at the translational level. Moreover, we found that RprA exerts negative feedback on the Cpx response, reducing Cpx activity in a manner that is dependent on the response regulator CpxR but independent of all of RprA's previously described targets. sRNAs therefore permit the fine-tuning of Cpx pathway activity and its regulation of target genes, which could assist bacterial survival in the face of envelope stress., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Gram-negative bacteria possess several envelope stress responses that detect and respond to damage to this critical cellular compartment. The σ(E) envelope stress response senses the misfolding of outer membrane proteins (OMPs), while the Cpx two-component system is believed to detect the misfolding of periplasmic and inner membrane proteins. Recent studies in several Gram-negative organisms found that deletion of hfq, encoding a small RNA chaperone protein, activates the σ(E) envelope stress response. In this study, we assessed the effects of deleting hfq upon activity of the σ(E) and Cpx responses in non-pathogenic and enteropathogenic (EPEC) strains of Escherichia coli. We found that the σ(E) response was activated in Δhfq mutants of all E. coli strains tested, resulting from the misregulation of OMPs. The Cpx response was activated by loss of hfq in EPEC, but not in E. coli K-12. Cpx pathway activation resulted in part from overexpression of the bundle-forming pilus (BFP) in EPEC Δhfq. We found that Hfq repressed expression of the BFP via PerA, a master regulator of virulence in EPEC. This study shows that Hfq has a more extensive role in regulating the expression of envelope proteins and horizontally acquired virulence genes in E. coli than previously recognized., (© 2014 John Wiley & Sons Ltd.)

Objective: Cervical cancer is a leading cause of cancer mortality in South Africa. However, little is known about oral human papillomavirus (HPV) infection in high human immunodeficiency virus (HIV) seroprevalence settings., Method: Thirty-four adult heterosexual couples attending an HIV testing center in Soweto, South Africa were enrolled. Each participant provided an oral rinse sample and genital swab, which were tested for 37 types of HPV DNA, and completed a risk behavior survey., Results: Median age was 31 years and 9% (3/34) of men and 29% (10/34) of women enrolled tested HIV-positive; median CD4 count was 437 cells/mm(3). Oral HPV prevalence was similar in women and men (12 vs. 18%, p = 0.48), and was non-significantly higher in HIV-infected vs. HIV-uninfected (23 vs. 13%, p = 0.34) subjects. Most men (82%) and women (84%) reported ever performing oral sex. Median number of lifetime sexual partners was "2-5" while median number of lifetime oral sex partners was 1. Oncogenic HPV subtypes were detected in 4% of oral, 26% of penile, and 74% of vaginal samples, including HPV16 in 1, 12, and 21% of these samples respectively. Genital HPV prevalence was significantly higher than oral HPV prevalence (75 vs. 15%, p ≤ 0.001). Thirty-five percent of couples (12/34) had at least one type-specific concordant vaginal-penile HPV infection but only one of nine couples with oral HPV had concordant oral-oral infection. However, 67% (4/6) of men and 25% (1/4) of women with oral HPV infection had partners with concordant genital HPV infection. Implications and Impact: Oral-oral HPV concordance between couples is low, but oral-genital and genital-genital HPV concordance is higher, including concordance of male oral HPV infection with their partners' vaginal HPV infection. This data is consistent with possible transmission of vaginal HPV infection to the oral cavity of sexual partners performing oral sex.

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.

The stringent response is a regulatory system that allows bacteria to sense and adapt to nutrient-poor environments. The central mediator of the stringent response is the molecule guanosine 3',5'-bispyrophosphate (ppGpp), which is synthesized by the enzymes RelA and SpoT and which is also degraded by SpoT. Our laboratory previously demonstrated that a relA mutant of Pseudomonas aeruginosa, the principal cause of lung infections in cystic fibrosis patients, was attenuated in virulence in a Drosophila melanogaster feeding model of infection. In this study, we examined the role of spoT in P. aeruginosa virulence. We generated an insertion mutation in spoT within the previously constructed relA mutant, thereby producing a ppGpp-devoid strain. The relA spoT double mutant was unable to establish a chronic infection in D. melanogaster and was also avirulent in the rat lung agar bead model of infection, a model in which the relA mutant is fully virulent. Synthesis of the virulence determinants pyocyanin, elastase, protease, and siderophores was impaired in the relA spoT double mutant. This mutant was also defective in swarming and twitching, but not in swimming motility. The relA spoT mutant and, to a lesser extent, the relA mutant were less able to withstand stresses such as heat shock and oxidative stress than the wild-type strain PAO1, which may partially account for the inability of the relA spoT mutant to successfully colonize the rat lung. Our results indicate that the stringent response, and SpoT in particular, is a crucial regulator of virulence processes in P. aeruginosa.

Background and Objectives: We characterized HCV antibody prevalence, viral persistence, genotype and liver disease prevalence among IDUs in Chennai, India as the study of the association of HIV with each of these states is important and there are no data available., Methods: Between 2005-2006, 1158 IDUs were recruited and followed semi-annually. All were tested for HCV antibodies at baseline; a random sample of 400 antibody positives (200 HIV-positive and 200 HIV-negative) were tested for HCV RNA; 13 of these were sequenced. Assessment of asparate amino transferase (AST)-to-platelet ratio index (APRI) was done on 557 IDUs. Prevalence ratios of each outcome were examined., Results: Median age was 35 yr; 99 per cent were male. HCV antibody prevalence was 55 per cent and was associated with older age, being unmarried, longer injection history, tattoo and injecting at a dealer's place. Of the 400 HCV antibody positive IDUs, 281 (70.3%) had persistent infection which was less common among hepatitis B-infected persons but not associated with HIV. Of the 13 samples sequenced, 11 (85%) were HCV genotype 3a. Fibrosis prevalence according to APRI was: HIV/HCV-uninfected, 4 per cent; HIV mono-infected, 3 per cent; HCV mono-infected, 11 per cent; HIV/HCV co-infected, 12 per cent (P<0.001). In addition to being associated with HCV and HIV/HCV, fibrosis prevalence was higher among those drinking alcohol frequently; daily marijuana use was protective., Interpretation and Conclusions: Our findings show that IDUs in Chennai have high HCV prevalence and associated disease burden. The burden will increase as access to antiretroviral therapy improves particularly given the high prevalence of HIV, HCV and alcohol use.

Enteropathogenic Escherichia coli (EPEC) are a major cause of infant morbidity and mortality due to diarrhoea in developing countries. The pathogenesis of EPEC is dependent on a coordinated multi-step process culminating in the intimate adherence of the organisms to the host's intestinal mucosa. During the initial stages of the EPEC colonization process, the fimbrial adhesin, bundle-forming pili (BFP), plays an integral role. We previously reported that the major BFP structural subunit, bundlin, displays lectin-like properties, which enables BFP to initially tether EPEC to N-acetyllactosamine (LacNAc) glycan receptors on host cell surfaces. We also reported that incubating EPEC with synthetic LacNAc-bearing neoglycoconjugates not only inhibits their adherence to host cells, but also induces BFP retraction and subsequent degradation of the bundlin subunits. Herein, we demonstrate that the periplasmic serine protease, DegP, is required for degrading bundlin during this process. We also show that DegP appears to act as a bundlin chaperone during BFP assembly and that LacNAc-BSA-induced BFP retraction is followed by transcriptional upregulation of the BFP operon and downregulation of the locus of enterocyte effacement operons in EPEC.

The Cpx envelope stress response is induced by the misfolding of periplasmic proteins and restores envelope homeostasis by upregulating several periplasmic protein folding and degrading factors. The Cpx response also regulates the expression of a variety of envelope-spanning protein complexes, including flagella, secretion systems and pili, which play an important role in pathogenesis. In a previous study, we inactivated the Cpx response in enteropathogenic Escherichia coli (EPEC), a causative agent of infant diarrhoea, and observed decreased expression of its major adhesin, the bundle-forming pilus (BFP). Here, we examined the mechanism underlying this BFP expression defect, and found that this phenotype can be attributed to insufficient expression of periplasmic folding factors, such as DsbA, DegP and CpxP. Hence, a low level of Cpx pathway activity promotes BFP synthesis by upregulating factors important for folding of BFP component proteins. Conversely, we found that full induction of the Cpx response inhibits BFP expression, mainly by repressing transcription of the bfp gene cluster. In combination with a previous report examining EPEC type III secretion, our results demonstrate that the Cpx response co-ordinates the repression of cell-surface structures during periods of envelope stress.