Bacterial-associated cholera toxin and GM 1 binding are required for transcytosis of classical biotype Vibrio cholerae through an in vitro M cell model system

To elucidate mechanisms involved in M cell uptake and transcytosis of Vibrio cholerae , we used an in vitro model of human M-like cells in a Caco-2 monolayer. Interspersed among the epithelial monolayer of Caco-2 cells we detect cells that display M-like features with or without prior lymphocyte treatment and we have established key parameters for V. cholerae transcytosis in this model. Cholera toxin (CT) mutants lacking the A subunit alone or both the A and B subunits were deficient for transcytosis. We explored this finding further and showed that expression of both subunits is required for binding by whole V. cholerae to immobilized CT receptor, the glycosphingolipid GM 1 . Confocal microscopy showed CT associated with transcytosing bacteria, and transcytosis was inhibited by pre-incubation with GM 1 before infection. Finally, heat treatment of the bacterial cells caused a loss of binding to GM 1 that was correlated with a significant decrease in uptake and transcytosis by the monolayer. Our data support a model in which the ability of bacteria to interact with GM 1 in a CT-dependent fashion plays a critical role in transcytosis of V. cholerae by M cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74960/1/j.1462-5822.2005.00681.x.pd

Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans in developed countries throughout the world. This bacterium frequently promotes a commensal lifestyle in the gastrointestinal tracts of many animals including birds and consumption or handling of poultry meats is a prevalent source of C. jejuni for infection in humans. To understand how the bacterium promotes commensalism, we used signature-tagged transposon mutagenesis and identified 29 mutants representing 22 different genes of C. jejuni strain 81–176 involved in colonization of the chick gastrointestinal tract. Among the determinants identified were two adjacent genes, one encoding a methyl-accepting chemotaxis protein (MCP), presumably required for proper chemotaxis to a specific environmental component, and another gene encoding a putative cytochrome c peroxidase that may function to reduce periplasmic hydrogen peroxide stress during in vivo growth. Deletion of either gene resulted in attenuation for growth throughout the gastrointestinal tract. Further examination of 10 other putative MCPs or MCP-domain containing proteins of C. jejuni revealed one other required for wild-type levels of caecal colonization. This study represents one of the first genetic screens focusing on the bacterial requirements necessary for promoting commensalism in a vertebrate host.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72403/1/j.1365-2958.2004.03988.x.pd

Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73261/1/j.1365-2958.2001.02376.x.pd

The PAS Domain-Containing Protein HeuR Regulates Heme Uptake in Campylobacter jejuni

Campylobacter jejuni is a leading cause of bacterially derived gastroenteritis. A previous mutant screen demonstrated that the heme uptake system (Chu) is required for full colonization of the chicken gastrointestinal tract. Subsequent work identified a PAS domain-containing regulator, termed HeuR, as being required for chicken colonization. Here we confirm that both the heme uptake system and HeuR are required for full chicken gastrointestinal tract colonization, with the hour mutant being particularly affected during competition with wild-type C. jejuni. Transcriptomic analysis identified the chu genes—and those encoding other iron uptake systems—as regulatory targets of HeuR. Purified HeuR bound the chuZA promoter region in electrophoretic mobility shift assays. Consistent with a role for HeuR in chu expression, heuR mutants were unable to efficiently use heme as a source of iron under iron-limiting conditions, and mutants exhibited decreased levels of cell-associated iron by mass spectrometry. Finally, we demonstrate that an heuR mutant of C. jejuni is resistant to hydrogen peroxide and that this resistance correlates to elevated levels of catalase activity. These results indicate that HeuR directly and positively regulates iron acquisition from heme and negatively impacts catalase activity by an as yet unidentified mechanism in C. jejuni

Differential Expression of the ToxR Regulon in Classical and E1 Tor Biotypes of Vibrio cholerae is Due to Biotype-Specific Control over toxT Expression.

The two major disease-causing biotypes of Vibrio cholerae, classical and El Tor, exhibit differences in their epidemic nature. Their behavior in the laboratory also differs in that El Tor strains produce two major virulence factors, cholera toxin (CT) and the toxin coregulated pilus (TCP), only under very restricted growth conditions, whereas classical strains do so in standard laboratory medium. Expression of toxin and TCP is controlled by two activator proteins, ToxR and ToxT, that operate in cascade fashion with ToxR controlling the synthesis of ToxT. Both biotypes express equivalent levels of ToxR, but only classical strains appear to express ToxT when grown in standard medium. In this report we show that restrictive expression of CT and TCP can be overcome in El Tor strains by expressing ToxT independently of ToxR. An El Tor strain lacking functional ToxT does not express CT or TCP, ruling out existence of a cryptic pathway for virulence regulation in this biotype. These results may have implications for understanding the evolution of El Tor strains toward reduced virulence with respect to classical strains

Single‐molecule tracking in live Vibrio cholerae reveals that ToxR recruits the membrane‐bound virulence regulator TcpP to the toxT promoter

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110815/1/mmi12834.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110815/2/mmi12834-sup-0001-si.pd

The Vibrio cholerae virulence regulatory cascade controls glucose uptake through activation of TarA, a small regulatory RNA

Vibrio cholerae causes the severe diarrhoeal disease cholera. A cascade of regulators controls expression of virulence determinants in V. cholerae at both transcriptional and post-transcriptional levels. ToxT is the direct transcription activator of the major virulence genes in V. cholerae . Here we describe TarA, a highly conserved, small regulatory RNA, whose transcription is activated by ToxT from toxboxes present upstream of the ToxT-activated gene tcpI . TarA regulates ptsG , encoding a major glucose transporter in V. cholerae . Cells overexpressing TarA exhibit decreased steady-state levels of ptsG mRNA and grow poorly in glucose-minimal media. A mutant lacking the ubiquitous regulatory protein Hfq expresses diminished TarA levels, indicating that TarA likely interacts with Hfq to regulate gene expression. RNAhybrid analysis of TarA and the putative ptsG mRNA leader suggests potential productive base-pairing between these two RNA molecules. A V. cholerae mutant lacking TarA is compromised for infant mouse colonization in competition with wild type, suggesting a role in the in vivo fitness of V. cholerae . Although somewhat functionally analogous to SgrS of Escherichia coli , TarA does not encode a regulatory peptide, and its expression is activated by the virulence gene pathway in V. cholerae and not by glycolytic intermediates.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79165/1/j.1365-2958.2010.07397.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/79165/2/MMI_7397_sm_TableS1.pd

Pharmaceutical applications of biotechnology: Promise and reality. Editorial overview

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30426/1/0000047.pd

Plasmonic nanoparticles assemblies templated by helical bacteria and resulting optical activity

Plasmonic nanoparticles (NPs) adsorbing onto helical bacteria can lead to formation of NP helicoids with micron scale pitch. Associated chiroptical effects can be utilized as bioanalytical tool for bacterial detection and better understanding of the spectral behavior of helical self‐assembled structures with different scales. Here, we report that enantiomerically pure helices with micron scale of chirality can be assembled on Campylobacter jejuni, a helical bacterium known for severe stomach infections. These organisms have right‐handed helical shapes with a pitch of 1–2 microns and can serve as versatile templates for a variety of NPs. The bacteria itself shows no observable rotatory activity in the visible, red, and near‐IR ranges of electromagnetic spectrum. The bacterial dispersion acquires chiroptical activity at 500–750 nm upon plasmonic functionalization with Au NPs. Finite‐difference time‐domain simulations confirmed the attribution of the chiroptical activity to the helical assembly of gold nanoparticles. The position of the circular dichroism peaks observed for these chiral structures overlaps with those obtained before for Au NPs and their constructs with molecular and nanoscale chirality. This work provides an experimental and computational pathway to utilize chiroplasmonic particles assembled on bacteria for bioanalytical purposes.Gold nanoparticles assemble onto the surface of helical bacterium, Campylobacter jejuni, producing right‐handed helices with a pitch of 1–2 microns. The bacterial dispersion acquires chiroptical activity at 500–750 nm that matches the calculated chiroplasmonic spectra. This study provides a pathway to utilize chiroplasmonic particles for monitoring shape dynamics of bacteria and identification of helical bacteria in complex microbiomes.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155927/1/Supporting_information_Chirality_Manuscript_2020.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155927/2/chir23225_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155927/3/chir23225.pd