bile-salt-induced-biofilm-formation-enteric-pathogens-techniques-for

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

epithelial-cell-infection-analyses-with-shigella

Epithelial Cell Infection Analyses with <em>Shigella</em>

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, ...

Massachusetts General Hospital

Harvard Medical School

OspF, OspG and IpaH(9.8) are type III secretion system (T3SS) effectors of Shigella flexneri that downregulate the host innate immune response. OspF modifies mitogen-activated protein kinase pathways and polymorphonuclear leucocyte transepithelial migration associated with Shigella invasion. OspF also localizes in the nucleus to mediate chromatin remodelling, resulting in reduced transcription of inflammatory cytokines. We now report that OspB can be added to the set of S. flexneri T3SS effectors required to modulate the innate immune response. T84 cells infected with a Delta ospB mutant resulted in reduced polymorphonuclear leucocyte transepithelial migration and mitogen-activated protein kinase signalling. Tagged versions of OspB localized with endosomes and the nucleus. Further, T84 cells infected with the Delta ospB mutant showed increased levels of secreted IL-8 compared with wild-type infected cells. Both GST-OspB and GST-OspF coprecipitated retinoblastoma protein from host cell lysates. Because Delta ospB and Delta ospF mutants share similar phenotypes, and OspB and OspF share a host binding partner, we propose that OspB and OspF facilitate the remodelling of chromatin via interactions with retinoblastoma protein, resulting in diminished inflammatory cytokine production. The requirement of multiple T3SS effectors to modulate the innate immune response correlates to the complexity of the human immune system.

Shigella flexneri type III secretion system effectors OspB and OspF target the nucleus to downregulate the host inflammatory response via interactions with retinoblastoma protein.

Bacterial pathogens have coevolved with humans in order to efficiently infect, replicate within, and be transmitted to new hosts to ensure survival and a continual infection cycle. For enteric pathogens, the ability to adapt to numerous host factors under the harsh conditions of the gastrointestinal tract is critical for establishing infection. One such host factor readily encountered by enteric bacteria is bile, an innately antimicrobial detergent-like compound essential for digestion and nutrient absorption. Not only have enteric pathogens evolved to resist the bactericidal conditions of bile, but these bacteria also utilize bile as a signal to enhance virulence regulation for efficient infection. This review provides a comprehensive and up-to-date analysis of bile-related research with enteric pathogens. From common responses to the unique expression of specific virulence factors, each pathogen has overcome significant challenges to establish infection in the gastrointestinal tract. Utilization of bile as a signal to modulate virulence factor expression has led to important insights for our understanding of virulence mechanisms for many pathogens. Further research on enteric pathogens exposed to this in vivo signal will benefit therapeutic and vaccine development and ultimately enhance our success at combating such elite pathogens.

Survival of the Fittest: How Bacterial Pathogens Utilize Bile To Enhance Infection.

Shigella flexneri is a Gram-negative pathogen that invades the colonic epithelium and causes millions of cases of watery diarrhea or bacillary dysentery predominately in children under the age of 5 years in developing countries. The effector Shigella enterotoxin 2 (ShET2), or OspD3, is encoded by the sen or ospD3 gene on the virulence plasmid. Previous literature has suggested that ospD3 is in an operon downstream of the ospC1 gene, and expression of both genes is controlled by a promoter upstream of ospC1. Since the intergenic region is 328 bases in length and contains several putative promoter regions, we hypothesized the genes are independently expressed. Here we provide data that ospD3 and ospC1 are not co-transcribed and that OspC1 is not required for OspD3/ShET2 function. Most importantly, we identified strong promoter activity in the intergenic region and demonstrate that OspD3/ShET2 can be expressed and secreted independently of OspC1. This work increases our understanding of the synthesis of a unique virulence factor and provides further insights into Shigella pathogenesis.

The synthesis of OspD3 (ShET2) in Shigella flexneri is independent of OspC1.

The  species cause millions of cases of watery or bloody diarrhea each year, mostly in children in developing countries. While many aspects of  colonic cell invasion are known, crucial gaps in knowledge regarding how the bacteria survive, transit, and regulate gene expression prior to infection remain. In this study, we define mechanisms of resistance to bile salts and build on previous research highlighting induced virulence in  strain 2457T following exposure to bile salts. Typical growth patterns were observed within the physiological range of bile salts; however, growth was inhibited at higher concentrations. Interestingly, extended periods of exposure to bile salts led to biofilm formation, a conserved phenotype that we observed among members of the  Characterization of  2457T biofilms determined that both bile salts and glucose were required for formation, dispersion was dependent upon bile salts depletion, and recovered bacteria displayed induced adherence to HT-29 cells. RNA-sequencing analysis verified an important bile salt transcriptional profile in  2457T, including induced drug resistance and virulence gene expression. Finally, functional mutagenesis identified the importance of the AcrAB efflux pump and lipopolysaccharide O-antigen synthesis for bile salt resistance. Our data demonstrate that  2457T employs multiple mechanisms to survive exposure to bile salts, which may have important implications for multidrug resistance. Furthermore, our work confirms that bile salts are important physiological signals to activate  2457T virulence. This work provides insights into how exposure to bile likely regulates  survival and virulence during host transit and subsequent colonic infection.

Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts.

Neutrophil influx into the intestinal lumen is a critical response to infectious agents, but is also associated with severe intestinal damage observed in idiopathic inflammatory bowel disease. The chemoattractant hepoxilin A3, an eicosanoid secreted from intestinal epithelial cells by the apically restricted efflux pump multidrug resistance protein 2 (MRP2), mediates this neutrophil influx. Information about a possible counterbalance pathway that could signal the lack of or resolution of an apical inflammatory signal, however, has yet to be described. We now report a system with such hallmarks. Specifically, we identify endocannabinoids as the first known endogenous substrates of the apically restricted multidrug resistance transporter P-glycoprotein (P-gp) and reveal a mechanism, which we believe is novel, for endocannabinoid secretion into the intestinal lumen. Knockdown or inhibition of P-gp reduced luminal secretion levels of N-acyl ethanolamine-type endocannabinoids, which correlated with increased neutrophil transmigration in vitro and in vivo. Additionally, loss of CB2, the peripheral cannabinoid receptor, led to increased pathology and neutrophil influx in models of acute intestinal inflammation. These results define a key role for epithelial cells in balancing the constitutive secretion of antiinflammatory lipids with the stimulated secretion of proinflammatory lipids via surface efflux pumps in order to control neutrophil infiltration into the intestinal lumen and maintain homeostasis in the healthy intestine.

Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis.

Enteric bacterial pathogens cause diarrheal disease and mortality at significant rates throughout the world, particularly in children younger than 5 years. Our ability to combat bacterial pathogens has been hindered by antibiotic resistance, a lack of effective vaccines, and accurate models of infection. With the renewed interest in bacteriophage therapy, we sought to use a novel human intestinal model to investigate the efficacy of a newly isolated bacteriophage against Shigella flexneri.

Bacteriophage Therapy Testing Against Shigella flexneri in a Novel Human Intestinal Organoid-Derived Infection Model.

The  species are Gram-negative, facultative intracellular pathogens that invade the colonic epithelium and cause significant diarrheal disease. Despite extensive research on the pathogen, a comprehensive understanding of how  initiates contact with epithelial cells remains unknown.  maintains many of the same  adherence gene operons; however, at least one critical gene component in each operon is currently annotated as a pseudogene in reference genomes. These annotations, coupled with a lack of structures upon microscopic analysis following growth in laboratory media, have led the field to hypothesize that  is unable to produce fimbriae or other traditional adherence factors. Nevertheless, our previous analyses have demonstrated that a combination of bile salts and glucose induces both biofilm formation and adherence to colonic epithelial cells. The goal of this study was to perform transcriptomic and genetic analyses to demonstrate that adherence gene operons in  strain 2457T are functional, despite the gene annotations. Our results demonstrate that at least three structural genes facilitate  2457T adherence for epithelial cell contact and biofilm formation. Furthermore, our results demonstrate that host factors, namely, glucose and bile salts at their physiological concentrations in the small intestine, offer key environmental stimuli required for adherence factor expression in  This research may have a significant impact on  vaccine development and further highlights the importance of utilizing -like conditions to study bacterial pathogenesis. Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The  species infect the epithelial cells lining the colon to result in millions of cases of diarrhea and a significant global health burden. As antibiotic resistance rates increase, understanding the mechanisms of infection is vital to ensure successful vaccine development. Despite significant gains in our understanding of  infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but  was thought to have lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some features of gastrointestinal transit and that enable  to express adherence structural genes. This work highlights aspects of genetic regulation for  adherence factors and may have a significant impact on future vaccine development.

 Adherence Factor Expression in -Like Conditions.

serovar Typhi is the etiologic agent of typhoid fever, a major public health problem in the developing world. Moving toward and adhering to the intestinal epithelium represents key initial steps of infection by . Typhi. We examined the role of the . Typhi  gene, which encodes an inner membrane phospholipid transporter, in these interactions with epithelial cells. Disruption of  resulted in elevated expression of flagellin and a hypermotile phenotype. It also significantly reduced the ability of . Typhi to adhere to the HeLa epithelial cell line and to polarized primary epithelial cells derived from human ileal organoids. Interestingly, the -deficient strain of . Typhi induced higher production of interleukin-8 from the primary human ileal epithelial cell monolayers compared to the wild-type bacteria. Deletion of the flagellin gene () in the -deficient . Typhi inhibited motility and attenuated interleukin-8 production, but it did not correct the defect in adhesion. We also disrupted  in . Typhimurium. In contrast to the results in . Typhi, the deficiency of  in . Typhimurium had no significant effect on flagellin expression, motility or adhesion to HeLa cells. Correspondingly, the lack of  also had no effect on association with the intestine or the severity of intestinal inflammation in the mouse model of . Typhimurium infection. Thus, our results point to an important and serovar-specific role played by  in the early stages of intestinal infection by . Typhi.

The YrbE phospholipid transporter of  serovar Typhi regulates the expression of flagellin and influences motility, adhesion and induction of epithelial inflammatory responses.

The molecular complexity of host-pathogen interactions remains poorly understood in many infectious diseases, particularly in humans due to the limited availability of reliable and specific experimental models. To bridge the gap between classical two-dimensional culture systems, which often involve transformed cell lines that may not have all the physiologic properties of primary cells, and in vivo animal studies, researchers have developed the organoid model system. Organoids are complex three-dimensional structures that are generated in vitro from primary cells and can recapitulate key in vivo properties of an organ such as structural organization, multicellularity, and function. In this review, we discuss how organoids have been deployed in exploring  infection in mice and humans. In addition, we summarize the recent advancements that hold promise to elevate our understanding of the interactions and crosstalk between multiple cell types and the microbiota with . These models have the potential for improving clinical outcomes and future prophylactic and therapeutic intervention strategies.

Spheres of Influence: Insights into  Pathogenesis from Intestinal Organoids.

Throughout the course of infection, many pathogens encounter bactericidal conditions that threaten the viability of the bacteria and impede the establishment of infection. Bile is one of the most innately bactericidal compounds present in humans, functioning to reduce the bacterial burden in the gastrointestinal tract while also aiding in digestion. It is becoming increasingly apparent that pathogens successfully resist the bactericidal conditions of bile, including bacteria that do not normally cause gastrointestinal infections. This review highlights the ability of , , ,  (ESKAPE), and other enteric pathogens to resist bile and how these interactions can impact the sensitivity of bacteria to various antimicrobial agents. Given that pathogen exposure to bile is an essential component to gastrointestinal transit that cannot be avoided, understanding how bile resistance mechanisms align with antimicrobial resistance is vital to our ability to develop new, successful therapeutics in an age of widespread and increasing antimicrobial resistance.

The Great ESKAPE: Exploring the Crossroads of Bile and Antibiotic Resistance in Bacterial Pathogens.

The mechanism of protection against cholera afforded by previous illness or vaccination is currently unknown. We have recently shown that antibodies targeting O-specific polysaccharide (OSP) of  correlate highly with protection against cholera.  is highly motile and possesses a flagellum sheathed in OSP, and motility of  correlates with virulence. Using high-speed video microscopy and building upon previous animal-related work, we demonstrate that sera, polyclonal antibody fractions, and OSP-specific monoclonal antibodies recovered from humans surviving cholera block  motility at both subagglutinating and agglutinating concentrations. This antimotility effect is reversed by preadsorbing sera and polyclonal antibody fractions with purified OSP and is associated with OSP-specific but not flagellin-specific monoclonal antibodies. Fab fragments of OSP-specific polyclonal antibodies do not inhibit motility, suggesting a requirement for antibody-mediated cross-linking in motility inhibition. We show that OSP-specific antibodies do not directly affect  viability, but that OSP-specific monoclonal antibody highly protects against death in the murine cholera model. We used  competitive index studies to demonstrate that OSP-specific antibodies impede colonization and survival of  in intestinal tissues and that this impact is motility dependent. Our findings suggest that the impedance of motility by antibodies targeting  OSP contributes to protection against cholera. Cholera is a severe dehydrating illness of humans caused by  is a highly motile bacterium that has a single flagellum covered in lipopolysaccharide (LPS) displaying O-specific polysaccharide (OSP), and  motility correlates with its ability to cause disease. The mechanisms of protection against cholera are not well understood; however, since  is a noninvasive intestinal pathogen, it is likely that antibodies that bind the pathogen or its products in the intestinal lumen contribute to protection from infection. Here, we demonstrate that OSP-specific antibodies isolated from humans surviving cholera in Bangladesh inhibit  motility and are associated with protection against challenge in a motility-dependent manner.

Humans Surviving Cholera Develop Antibodies against Vibrio cholerae O-Specific Polysaccharide That Inhibit Pathogen Motility.

Gastrointestinal infections cause significant morbidity and mortality worldwide. The complexity of human biology and limited insights into host-specific infection mechanisms are key barriers to current therapeutic development. Here, we demonstrate that two-dimensional epithelial monolayers derived from human intestinal organoids, combined with like bacterial culturing conditions, provide significant advancements for the study of enteropathogens. Monolayers from the terminal ileum, cecum, and ascending colon recapitulated the composition of the gastrointestinal epithelium, in which several techniques were used to detect the presence of enterocytes, mucus-producing goblet cells, and other cell types following differentiation. Importantly, the addition of receptor activator of nuclear factor kappa-B ligand (RANKL) increased the presence of M cells, critical antigen-sampling cells often exploited by enteric pathogens. For infections, bacteria were grown under like conditions known to induce virulence. Overall, interesting patterns of tissue tropism and clinical manifestations were observed. Shigella flexneri adhered efficiently to the cecum and colon; however, invasion in the colon was best following RANKL treatment. Both Salmonella enterica serovars Typhi and Typhimurium displayed different infection patterns, with . Typhimurium causing more destruction of the terminal ileum and . Typhi infecting the cecum more efficiently than the ileum, particularly with regard to adherence. Finally, various pathovars of Escherichia coli validated the model by confirming only adherence was observed with these strains. This work demonstrates that the combination of human-derived tissue with targeted bacterial growth conditions enables powerful analyses of human-specific infections that could lead to important insights into pathogenesis and accelerate future vaccine development.  While traditional laboratory techniques and animal models have provided valuable knowledge in discerning virulence mechanisms of enteric pathogens, the complexity of the human gastrointestinal tract has hindered our understanding of physiologically relevant, human-specific interactions; and thus, has significantly delayed successful vaccine development. The human intestinal organoid-derived epithelial monolayer (HIODEM) model closely recapitulates the diverse cell populations of the intestine, allowing for the study of human-specific infections. Differentiation conditions permit the expansion of various cell populations, including M cells that are vital to immune recognition and the establishment of infection by some bacteria. We provide details of reproducible culture methods and infection conditions for the analyses of , , and pathogenic Escherichia coli in which tissue tropism and pathogen-specific infection patterns were detected. This system will be vital for future studies that explore infection conditions, health status, or epigenetic differences and will serve as a novel screening platform for therapeutic development.

A Versatile Human Intestinal Organoid-Derived Epithelial Monolayer Model for the Study of Enteric Pathogens.

Alarming rates of antibiotic resistance in bacteria and gastrointestinal dysbiosis associated with traditional antimicrobial therapy have led to renewed interests in developing bacteriophages as novel therapeutics. In this review, we highlight some of the recent advances in bacteriophage therapeutic development targeting important enteropathogens of the gastrointestinal tract.

Bacteriophages against enteropathogens: rediscovery and refinement of novel antimicrobial therapeutics.

The gastrointestinal (GI) epithelium plays a major role in nutrient absorption, barrier formation, and innate immunity. The development of organoid-based methodology has significantly impacted the study of the GI epithelium, particularly in the fields of mucosal biology, immunity, and host-microbe interactions. Various effects on the GI epithelium, such as genetics and nutrition, impact patients and alter disease states. Thus, incorporating these effects into organoid-based models will facilitate a better understanding of disease progression and offer opportunities to evaluate therapeutic candidates. One condition that has a significant effect on the GI epithelium is malnutrition, and studying the mechanistic impacts of malnutrition would enhance our understanding of several pathologies. Therefore, the goal of this study was to begin to develop methodology to generate viable malnourished organoids with accessible techniques and resources that can be used for a wide array of mechanistic studies. By selectively limiting distinct macronutrient components of organoid media, we were able to successfully culture and evaluate malnourished organoids. Genetic and protein-based analyses were used to validate the approach and confirm the presence of known biomarkers of malnutrition. Additionally, as proof-of-concept, we utilized malnourished organoid-derived monolayers to evaluate the effect of malnourishment on barrier formation and the ability of the bacterial pathogen  to infect the GI epithelium. This work serves as the basis for new and exciting techniques to alter the nutritional state of organoids and investigate the related impacts on the GI epithelium.

A foundational approach to culture and analyze malnourished organoids.

We have identified a decreased abundance of microbial species known to have a potential anti-inflammatory, protective effect in subjects that developed Celiac Disease (CeD) compared to those who did not. We aim to confirm the potential protective role of one of these species, namely Bacteroides vulgatus, and to mechanistically establish the effect of bacterial bioproducts on gluten-dependent changes on human gut epithelial functions.

Novel Bacteroides Vulgatus strain protects against gluten-induced break of human celiac gut epithelial homeostasis: a pre-clinical proof-of-concept study.

ArticleTotal : 2	Year
Publication title Cited by 18	2018
Publication title Cited by 1	2024

Article	Year
Shigella flexneri type III secretion system effectors OspB and OspF target the nucleus to downregulate the host inflammatory response via interactions with retinoblastoma protein. Molecular microbiology\| PubMed ID: 19017275	2009
Survival of the Fittest: How Bacterial Pathogens Utilize Bile To Enhance Infection. Clinical microbiology reviews\| PubMed ID: 27464994	2016
The synthesis of OspD3 (ShET2) in Shigella flexneri is independent of OspC1. Gut microbes\| PubMed ID: 27657187	2016
Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts. Infection and immunity\| PubMed ID: 28348056	2017
Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis. The Journal of clinical investigation\| PubMed ID: 30102254	2018
Bacteriophage Therapy Testing Against Shigella flexneri in a Novel Human Intestinal Organoid-Derived Infection Model. Journal of pediatric gastroenterology and nutrition\| PubMed ID: 30418409	2019
Adherence Factor Expression in -Like Conditions. mSphere\| PubMed ID: 31722995	2019
The YrbE phospholipid transporter of serovar Typhi regulates the expression of flagellin and influences motility, adhesion and induction of epithelial inflammatory responses. Gut microbes\| PubMed ID: 31829769	2020
Spheres of Influence: Insights into Pathogenesis from Intestinal Organoids. Microorganisms\| PubMed ID: 32244707	2020
The Great ESKAPE: Exploring the Crossroads of Bile and Antibiotic Resistance in Bacterial Pathogens. Infection and immunity\| PubMed ID: 32661122	2020
Humans Surviving Cholera Develop Antibodies against Vibrio cholerae O-Specific Polysaccharide That Inhibit Pathogen Motility. mBio\| PubMed ID: 33203761	2020
A Versatile Human Intestinal Organoid-Derived Epithelial Monolayer Model for the Study of Enteric Pathogens. Microbiology spectrum\| PubMed ID: 34106568	2021
Bacteriophages against enteropathogens: rediscovery and refinement of novel antimicrobial therapeutics. Current opinion in infectious diseases\| PubMed ID: 34524200	2021
A foundational approach to culture and analyze malnourished organoids. Gut microbes\| PubMed ID: 37724815	2023
Novel Bacteroides Vulgatus strain protects against gluten-induced break of human celiac gut epithelial homeostasis: a pre-clinical proof-of-concept study. Pediatric research\| PubMed ID: 38177249	2024

Christina S. Faherty

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