scholarly journals Shigella flexneri Adherence Factor Expression in In Vivo-Like Conditions

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Rachael B. Chanin ◽  
Kourtney P. Nickerson ◽  
Alejandro Llanos-Chea ◽  
Jeticia R. Sistrunk ◽  
David A. Rasko ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Biofilm Formation ◽  
Bile Salts ◽  
Vaccine Development ◽  
Shigella Flexneri ◽  
Colonic Epithelium ◽  
Structural Genes ◽  
Content Type ◽  
Factor Expression

ABSTRACT The Shigella 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 Shigella initiates contact with epithelial cells remains unknown. Shigella maintains many of the same Escherichia coli 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 Shigella 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 Shigella flexneri strain 2457T are functional, despite the gene annotations. Our results demonstrate that at least three structural genes facilitate S. flexneri 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 S. flexneri. This research may have a significant impact on Shigella vaccine development and further highlights the importance of utilizing in vivo-like conditions to study bacterial pathogenesis. IMPORTANCE Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The Shigella 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 Shigella infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but Shigella 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 Shigella to express adherence structural genes. This work highlights aspects of genetic regulation for Shigella adherence factors and may have a significant impact on future vaccine development.

scholarly journals Shigella flexneri adherence factor expression in in vivo-like conditions

2019 ◽  
Author(s):  
Rachael B. Chanin ◽  
Kourtney P. Nickerson ◽  
Alejandro Llanos-Chea ◽  
Jeticia R. Sistrunk ◽  
David A. Rasko ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Biofilm Formation ◽  
Bile Salts ◽  
Diarrheal Disease ◽  
Vaccine Development ◽  
Gastrointestinal Transit ◽  
Cell Contact ◽  
Colonic Epithelium ◽  
Factor Expression

AbstractThe Shigella species are Gram-negative, facultative intracellular pathogens that invade the colonic epithelium and cause significant diarrheal disease. Despite extensive research on the pathogen, comprehensive understanding of how Shigella initiates contact with epithelial cells remains unknown. Shigella maintains many of the same Escherichia coli 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 Shigella is unable to produce fimbriae or other “traditional” adherence factors. Nevertheless, our previous analyses have demonstrated that a combination of bile salts and glucose induce both biofilm formation and adherence to colonic epithelial cells. Through a two-part investigation, we first utilized various transcriptomic analyses to demonstrate that S. flexneri strain 2457T adherence gene operons are transcribed. Subsequently, we performed mutation, electron microscopy, biofilm, infection, and proteomic analyses to characterize three of the structural genes. In combination, these studies demonstrate that despite the gene annotations, S. flexneri 2457T uses adherence factors to initiate biofilm formation as well as epithelial cell contact. Furthermore, host factors, namely glucose and bile salts in the small intestine, offer key environmental stimuli required for proper adherence factor expression in S. flexneri. This research may have a significant impact on vaccine development for Shigella and further highlights the importance of utilizing in vivo-like conditions to study bacterial pathogenesis.ImportanceBacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The Shigella 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 are vital to ensure successful vaccine development. Despite significant gains in our understanding of Shigella infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but Shigella was thought to have lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some features of gastrointestinal transit and enable Shigella to express adherence factors. This work highlights aspects of genetic regulation for Shigella adherence factors and may have a significant impact on future vaccine development.

scholarly journals Pneumococcal Interactions with Epithelial Cells Are Crucial for Optimal Biofilm Formation and ColonizationIn VitroandIn Vivo

2012 ◽  
Vol 80 (8) ◽  
pp. 2744-2760 ◽  
Author(s):  
Laura R. Marks ◽  
G. Iyer Parameswaran ◽  
Anders P. Hakansson
Keyword(s):  
Epithelial Cells ◽  
Biofilm Formation ◽  
Invasive Disease ◽  
Respiratory Epithelial Cells ◽  
Symptomatic Infection ◽  
Content Type ◽  
Abiotic Surfaces ◽  
Horizontal Spread ◽  
Respiratory Epithelial

ABSTRACTThe human nasopharynx is the main reservoir forStreptococcus pneumoniae(the pneumococcus) and the source for both horizontal spread and transition to infection. Some clinical evidence indicates that nasopharyngeal carriage is harder to eradicate with antibiotics than is pneumococcal invasive disease, which may suggest that colonizing pneumococci exist in biofilm communities that are more resistant to antibiotics. While pneumococcal biofilms have been observed during symptomatic infection, their role in colonization and the role of host factors in this process have been less studied. Here, we show for the first time that pneumococci form highly structured biofilm communities during colonization of the murine nasopharynx that display increased antibiotic resistance. Furthermore, pneumococcal biofilms grown on respiratory epithelial cells exhibited phenotypes similar to those observed during colonizationin vivo, whereas abiotic surfaces produced less ordered and more antibiotic-sensitive biofilms. The importance of bacterial-epithelial cell interactions during biofilm formation was shown using both clinical strains with variable colonization efficacies and pneumococcal mutants with impaired colonization characteristicsin vivo. In both cases, the ability of strains to form biofilms on epithelial cells directly correlated with their ability to colonize the nasopharynxin vivo, with colonization-deficient strains forming less structured and more antibiotic-sensitive biofilms on epithelial cells, an association that was lost when grown on abiotic surfaces. Thus, these studies emphasize the importance of host-bacterial interactions in pneumococcal biofilm formation and provide the first experimental data to explain the high resistance of pneumococcal colonization to eradication by antibiotics.

scholarly journals Identification of Salmonella enterica Serovar Typhimurium Genes Regulated during Biofilm Formation on Cholesterol Gallstone Surfaces

2013 ◽  
Vol 81 (10) ◽  
pp. 3770-3780 ◽  
Author(s):  
Geoffrey Gonzalez-Escobedo ◽  
John S. Gunn
Keyword(s):  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Cholesterol Gallstone ◽  
Structural Genes ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Coated Surfaces ◽  
Biofilm Development

ABSTRACTSalmonellaspp. are able to form biofilms on abiotic and biotic surfaces.In vivostudies in our laboratory have shown thatSalmonellacan form biofilms on the surfaces of cholesterol gallstones in the gallbladders of mice and human carriers. Biofilm formation on gallstones has been demonstrated to be a mechanism of persistence. The purpose of this work was to identify and evaluateSalmonellasp. cholesterol-dependent biofilm factors. Differential gene expression analysis between biofilms on glass or cholesterol-coated surfaces and subsequent quantitative real-time PCR (qRT-PCR) revealed that type 1 fimbria structural genes and a gene encoding a putative outer membrane protein (ycfR) were specifically upregulated inSalmonella entericaserovar Typhimurium biofilms grown on cholesterol-coated surfaces. Spatiotemporal expression ofycfRand FimA verified their regulation during biofilm development on cholesterol-coated surfaces. Surprisingly, confocal and scanning electron microscopy demonstrated that a mutant of type 1 fimbria structural genes (ΔfimAICDHF) and aycfRmutant showed increased biofilm formation on cholesterol-coated surfaces.In vivoexperiments usingNramp1+/+mice harboring gallstones showed that only the ΔycfRmutant formed extensive biofilms on mouse gallstones at 7 and 21 days postinfection; ΔfimAICDHFwas not observed on gallstone surfaces after the 7-day-postinfection time point. These data suggest that inSalmonellaspp., wild-type type 1 fimbriae are important for attachment to and/or persistence on gallstones at later points of chronic infection, whereas YcfR may represent a specific potential natural inhibitor of initial biofilm formation on gallstones.

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

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Kourtney P. Nickerson ◽  
Rachael B. Chanin ◽  
Jeticia R. Sistrunk ◽  
David A. Rasko ◽  
Peter J. Fink ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bile Salt ◽  
Biofilm Formation ◽  
Bile Salts ◽  
Efflux Pump ◽  
Shigella Flexneri ◽  
Growth Patterns ◽  
Transcriptional Profile ◽  
Sequencing Analysis ◽  
Content Type

ABSTRACT The Shigella species cause millions of cases of watery or bloody diarrhea each year, mostly in children in developing countries. While many aspects of Shigella 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 Shigella flexneri 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 Enterobacteriaceae. Characterization of S. flexneri 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 S. flexneri 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 S. flexneri 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 S. flexneri 2457T virulence. This work provides insights into how exposure to bile likely regulates Shigella survival and virulence during host transit and subsequent colonic infection.

scholarly journals The Autotransporter IcsA PromotesShigella flexneriBiofilm Formation in the Presence of Bile Salts

2019 ◽  
Vol 87 (7) ◽  
Author(s):  
Volkan K. Köseoğlu ◽  
Chelsea P. Hall ◽  
Eric M. Rodríguez-López ◽  
Hervé Agaisse
Keyword(s):  
Biofilm Formation ◽  
Bile Salts ◽  
Colonic Mucosa ◽  
Shigella Flexneri ◽  
Bacterial Pathogen ◽  
Phenotypic Analysis ◽  
Content Type ◽  
Mutant Strains ◽  
Aggregative Growth

ABSTRACTShigella flexneriis an intracellular bacterial pathogen that invades epithelial cells in the colonic mucosa, leading to bloody diarrhea. A previous study showed thatS. flexneriforms biofilms in the presence of bile salts, through an unknown mechanism. Here, we investigated the potential role of adhesin-like autotransporter proteins inS. flexneribiofilm formation. BLAST search analysis revealed that theS. flexneri2457T genome harbors 4 genes,S1242,S1289,S2406, andicsA, encoding adhesin-like autotransporter proteins. Deletion mutants of theS1242,S1289,S2406andicsAgenes were generated and tested for biofilm formation. Phenotypic analysis of the mutant strains revealed that disruption oficsAabolished bile salt-induced biofilm formation. IcsA is an outer membrane protein secreted at the bacterial pole that is required forS. flexneriactin-based motility during intracellular infection. In extracellular biofilms, IcsA was also secreted at the bacterial pole and mediated bacterial cell-cell contacts and aggregative growth in the presence of bile salts. Dissecting individual roles of bile salts showed that deoxycholate is a robust biofilm inducer compared to cholate. The release of the extracellular domain of IcsA through IcsP-mediated cleavage was greater in the presence of cholate, suggesting that the robustness of biofilm formation was inversely correlated with IcsA processing. Accordingly, deletion oficsPabrogated IcsA processing in biofilms and enhanced biofilm formation.

scholarly journals Gallbladder Epithelium as a Niche for Chronic Salmonella Carriage

2013 ◽  
Vol 81 (8) ◽  
pp. 2920-2930 ◽  
Author(s):  
Geoffrey Gonzalez-Escobedo ◽  
John S. Gunn
Keyword(s):  
Epithelial Cells ◽  
Biofilm Formation ◽  
Dependent Manner ◽  
Gallbladder Epithelium ◽  
Content Type ◽  
Bacterial Aggregation ◽  
Chronic Carriage ◽  
Cell Extrusion

ABSTRACTAlthough typhoid fever has been intensively studied, chronic typhoid carriage still represents a problem for the transmission and persistence of the disease in areas of endemicity. This chronic state is highly associated with the presence of gallstones in the gallbladder of infected carriers upon whichSalmonellacan form robust biofilms. However, we hypothesize that in addition to gallstones, the gallbladder epithelium aids in the establishment/maintenance of chronic carriage. In this work, we present evidence of the role of the gallbladder epithelium in chronic carriage by a mechanism involving invasion, intracellular persistence, and biofilm formation.Salmonellawas able to adhere to and invade polarized gallbladder epithelial cells apically in the absence and presence of bile in aSalmonellapathogenicity island 1 (SPI-1)-dependent manner. Intracellular replication ofSalmonellawas also evident at 12 and 24 h postinvasion. A flowthrough system revealed thatSalmonellais able to adhere to and form extensive bacterial foci on gallbladder epithelial cells as early as 12 h postinoculation.In vivoexperiments using a chronic mouse model of typhoid carriage showed invasion and damage of the gallbladder epithelium and lamina propria up to 2 months afterSalmonellainfection, with an abundant presence of macrophages, a relative absence of neutrophils, and extrusion of infected epithelial cells. Additionally, microcolonies ofSalmonellacells were evident on the surface of the mouse gallbladder epithelia up to 21 days postinfection. These data reveal a second potential mechanism, intracellular persistence and/or bacterial aggregation in/on the gallbladder epithelium with luminal cell extrusion, forSalmonellamaintenance in the gallbladder.

scholarly journals Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

2011 ◽  
Vol 80 (1) ◽  
pp. 3-13 ◽  
Author(s):  
Chen Li ◽  
Kurniyati ◽  
Bo Hu ◽  
Jiang Bian ◽  
Jianlan Sun ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Biofilm Formation ◽  
Adult Population ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Content Type ◽  
Multiple Organs ◽  
Biochemical Analyses

ABSTRACTThe oral bacteriumPorphyromonas gingivalisis a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide.P. gingivalisexhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence ofP. gingivalisremain elusive. In this report, we found thatP. gingivalisencodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed thatPG0352is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPgis an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that thePG0352deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type,in vitrostudies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement.In vivostudies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPgis an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity ofP. gingivalis, and it can potentially serve as a new target for developing therapeutic agents againstP. gingivalisinfection.

scholarly journals The Combinations Chitosan-Pam3CSK4 and Chitosan-Monophosphoryl Lipid A: Promising Immune-Enhancing Adjuvants for Anticaries Vaccine PAc

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Yongli Bi ◽  
Qingan Xu ◽  
Lingkai Su ◽  
Jiantao Xu ◽  
Zhongfang Liu ◽  
...  
Keyword(s):  
Lipid A ◽  
Vaccine Development ◽  
Protection Effect ◽  
Content Type ◽  
Monophosphoryl Lipid A ◽  
Monophosphoryl Lipid ◽  
Tooth Surfaces ◽  
Antibody Titers

ABSTRACT We previously demonstrated that recombinant protein PAc could be administered as an anticaries vaccine. However, the relatively weak immunogenicity of PAc limits its application. In the present study, we investigated the effect of two adjuvant combinations of chitosan plus Pam3CSK4 (chitosan-Pam3CSK4) and of chitosan plus monophosphoryl lipid A (chitosan-MPL) in the immune responses to the PAc protein in vivo and in vitro. PAc-chitosan-Pam3CSK4 or PAc-chitosan-MPL promoted significantly higher PAc-specific antibody titers in serum and saliva, inhibited Streptococcus mutans colonization onto the tooth surfaces, and endowed better protection effect with significantly less caries activities than PAc alone. Chitosan-Pam3CSK4 and chitosan-MPL showed no statistically significant differences. In conclusion, our study demonstrated that the chitosan-Pam3CSK4 and chitosan-MPL combinations are promising for anticaries vaccine development.

scholarly journals Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

2012 ◽  
Vol 19 (10) ◽  
pp. 1603-1608 ◽  
Author(s):  
Koushik Roy ◽  
David J. Hamilton ◽  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Diarrheal Disease ◽  
Vaccine Development ◽  
Enterotoxigenic Escherichia Coli ◽  
Intestinal Colonization ◽  
Content Type ◽  
Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonizationin vivoand toxin delivery to epithelial cellsin vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development.

scholarly journals Evaluation of Antibiotics Active against Methicillin-Resistant Staphylococcus aureus Based on Activity in an Established Biofilm

2016 ◽  
Vol 60 (10) ◽  
pp. 5688-5694 ◽  
Author(s):  
Daniel G. Meeker ◽  
Karen E. Beenken ◽  
Weston B. Mills ◽  
Allister J. Loughran ◽  
Horace J. Spencer ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Limit Of Detection ◽  
Relative Activity ◽  
Methicillin Resistant ◽  
Content Type ◽  
Comparable Activity ◽  
Antibiotic Delivery

ABSTRACTWe usedin vitroandin vivomodels of catheter-associated biofilm formation to compare the relative activity of antibiotics effective against methicillin-resistantStaphylococcus aureus(MRSA) in the specific context of an established biofilm. The results demonstrated that, underin vitroconditions, daptomycin and ceftaroline exhibited comparable activity relative to each other and greater activity than vancomycin, telavancin, oritavancin, dalbavancin, or tigecycline. This was true when assessed using established biofilms formed by the USA300 methicillin-resistant strain LAC and the USA200 methicillin-sensitive strain UAMS-1. Oxacillin exhibited greater activity against UAMS-1 than LAC, as would be expected, since LAC is an MRSA strain. However, the activity of oxacillin was less than that of daptomycin and ceftaroline even against UAMS-1. Among the lipoglycopeptides, telavancin exhibited the greatest overall activity. Specifically, telavancin exhibited greater activity than oritavancin or dalbavancin when tested against biofilms formed by LAC and was the only lipoglycopeptide capable of reducing the number of viable bacteria below the limit of detection. With biofilms formed by UAMS-1, telavancin and dalbavancin exhibited comparable activity relative to each other and greater activity than oritavancin. Importantly, ceftaroline was the only antibiotic that exhibited greater activity than vancomycin when testedin vivoin a murine model of catheter-associated biofilm formation. These results emphasize the need to consider antibiotics other than vancomycin, most notably, ceftaroline, for the treatment of biofilm-associatedS. aureusinfections, including by the matrix-based antibiotic delivery methods often employed for local antibiotic delivery in the treatment of these infections.

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