Interactions between chaperone and energy storage networks during the evolution of Legionella pneumophila under heat shock.

Waterborne transmission of the bacterium Legionella pneumophila has emerged as a major cause of severe nosocomial infections of major public health impact. The major route of transmission involves the uptake of aerosolized bacteria, often from the contaminated hot water systems of large buildings. Public health regulations aimed at controlling the mesophilic pathogen are generally concerned with acute pasteurization and maintaining high temperatures at the heating systems and throughout the plumbing of hot water systems, but L. pneumophila is often able to survive these treatments due to both bacterium-intrinsic and environmental factors. Previous work has established an experimental evolution system to model the observations of increased heat resistance in repeatedly but unsuccessfully pasteurized L. pneumophila populations. Here, we show rapid fixation of novel alleles in lineages selected for resistance to heat shock and shifts in mutational profile related to increases in the temperature of selection. Gene-level and nucleotide-level parallelisms between independently-evolving lineages show the centrality of the DnaJ/DnaK chaperone system in the heat resistance of L. pneumophila. Inference of epistatic interactions through reverse genetics shows an unexpected interaction between DnaJ/DnaK and the polyhydroxybutyrate-accumulation energy storage mechanism used by the species to survive long-term starvation in low-nutrient environments.

Draft genome sequences of 13 Salmonella enterica subsp. enterica isolates from chickens, cows, and Canadian Salmonella outbreaks.

Salmonella enterica is the etiological agent responsible for salmonellosis. Here, we report the draft whole genome sequences of 13 S. enterica subsp. enterica isolates from chickens and cows, as well as from previous Canadian Salmonella outbreaks investigated by the Canadian Food Inspection Agency.

Development of heat-shock resistance in Legionella pneumophila modeled by experimental evolution.

Because it can grow in buildings with complex hot water distribution systems (HWDS), healthcare facilities recognize the waterborne bacterium Legionella pneumophila as a major nosocomial infection threat and often try to clear the systems with a pasteurization process known as superheat-and-flush. After this treatment, many facilities find that the contaminating populations slowly recover, suggesting the possibility of in situ evolution favoring increased survival in high-temperature conditions. To mimic this process in a controlled environment, an adaptive laboratory evolution model was used to select a wild-type strain of L. pneumophila for survival to transient exposures to temperatures characteristic of routine hot water use or failed pasteurization processes in HWDS. Over their evolution, these populations became insensitive to exposure to 55°C and developed the ability to survive short exposures to 59°C heat shock. Heat-adapted lineages maintained a higher expression of heat-shock genes during low-temperature incubation in freshwater, suggesting a pre-adaptation to heat stress. Although there were distinct mutation profiles in each of the heat-adapted lineages, each acquired multiple mutations in the DnaJ/DnaK/ClpB disaggregase complex, as well as mutations in chaperone htpG and protease clpX. These mutations were specific to heat-shock survival and were not seen in control lineages included in the experimental model without exposure to heat shock. This study supports in situ observations of adaptation to heat stress and demonstrates the potential of L. pneumophila to develop resistance to control measures. IMPORTANCE As a bacterium that thrives in warm water ecosystems, Legionella pneumophila is a key factor motivating regulations on hot water systems. Two major measures to control Legionella are high circulating temperatures intended to curtail growth and the use of superheat-and-flush pasteurization processes to eliminate established populations. Facilities often suffer recolonization of their hot water systems; hospitals are particularly at risk due to the severe nosocomial pneumoniae caused by Legionella. To understand these long-term survivors, we have used an adaptive laboratory evolution model to replicate this process. We find major differences between the mutational profiles of heat-adapted and heat-naïve L. pneumophila populations including mutations in major heat-shock genes like chaperones and proteases. This model demonstrates that well-validated treatment protocols are needed to clear contaminated systems and-in an analog to antibiotic resistance-the importance of complete eradication of the resident population to prevent selection for more persistent bacteria.

A tail-specific protease is required for Legionella pneumophila intracellular multiplication.

Legionella pneumophila is a Gram-negative bacterium found in natural and man-made water systems where it replicates within amoebas and ciliates. In humans, once inside the lungs, L. pneumophila replicates in alveolar macrophages and causes Legionnaires' disease, a severe pneumonia. The Icm/Dot type IVb secretion system is a major virulence factor required for intracellular multiplication. The Icm/Dot system allows the secretion of effectors into the cytoplasm of the host cell. These effectors modify host cell vesicular trafficking and prevent maturation of the phagosome. The innate immune response is crucial in restricting L. pneumophila proliferation. TNF-α is one of the major cytokines involved in this process as it renders macrophages more resistant to L. pneumophila infection and induces apoptosis of L. pneumophila-infected macrophages. Tail-specific proteases (Tsp) are involved in tolerating thermal stress and in virulence. We have previously characterized the Tsp encoded by L. pneumophila, showing that it is important for surviving thermal stress and for infection of amoeba when a temperature change occurs during infection. Here, we demonstrated that Tsp is required for intracellular multiplication in macrophages. Absence of tsp is associated with higher production of TNF-α by macrophages in response to L. pneumophila infection. This effect is independent of the Icm/Dot secretion system.

Toxoflavin secreted by Pseudomonas alcaliphila inhibits the growth of Legionella pneumophila and Vermamoeba vermiformis.

Legionella pneumophila is a natural inhabitant of water systems. From there, it can be transmitted to humans by aerosolization resulting in severe pneumonia. Most large outbreaks are caused by cooling towers colonized with L. pneumophila. The resident microbiota of the cooling tower is a key determinant for the colonization and growth of L. pneumophila. In our preceding study, the genus Pseudomonas correlated negatively with the presence of L. pneumophila in cooling towers, but it was not clear which species was responsible. Therefore, we identified the Pseudomonas species inhabiting 14 cooling towers using a Pseudomonas-specific 16S rRNA amplicon sequencing strategy. We found that cooling towers that are free of L. pneumophila contained a high relative abundance of members from the Pseudomonas alcaliphila/oleovorans phylogenetic cluster. P. alcaliphila JCM 10630 inhibited the growth of L. pneumophila on agar plates. Analysis of the P. alcaliphila genome revealed the presence of a gene cluster predicted to produce toxoflavin. L. pneumophila growth was inhibited by pure toxoflavin and by extracts from P. alcaliphila culture found to contain toxoflavin by liquid chromatography coupled with mass spectrometry. In addition, toxoflavin inhibits the growth of Vermameoba vermiformis, a host cell of L. pneumophila. Our study indicates that P. alcaliphila may be important to restrict growth of L. pneumophila in water systems through the production of toxoflavin. A sufficiently high concentration of toxoflavin is likely not achieved in the bulk water but might have a local inhibitory effect such as near or in biofilms.

Bacterial Antagonistic Species of the Pathogenic Genus Legionella Isolated from Cooling Tower.

Legionella pneumophila is the causative agent of Legionnaires' disease, a severe pneumonia. Cooling towers are a major source of large outbreaks of the disease. The growth of L. pneumophila in these habitats is influenced by the resident microbiota. Consequently, the aim of this study was to isolate and characterize bacterial species from cooling towers capable of inhibiting several strains of L. pneumophila and one strain of L. quinlivanii. Two cooling towers were sampled to isolate inhibiting bacterial species. Seven inhibitory isolates were isolated, through serial dilution plating and streaking on agar plates, belonging to seven distinct species. The genomes of these isolates were sequenced to identify potential genetic elements that could explain the inhibitory effect. The results showed that the bacterial isolates were taxonomically diverse and that one of the isolates may be a novel species. Genome analysis showed a high diversity of antimicrobial gene products identified in the genomes of the bacterial isolates. Finally, testing different strains of Legionella demonstrated varying degrees of susceptibility to the antimicrobial activity of the antagonistic species. This may be due to genetic variability between the Legionella strains. The results demonstrate that though cooling towers are breeding grounds for L. pneumophila, the bacteria must contend with various antagonistic species. Potentially, these species could be used to create an inhospitable environment for L. pneumophila, and thus decrease the probability of outbreaks occurring.

Aptamers and Aptamer-Coupled Biosensors to Detect Water-Borne Pathogens.

Aptamers can serve as efficient bioreceptors for the development of biosensing detection platforms. Aptamers are short DNA or RNA oligonucleotides that fold into specific structures, which enable them to selectively bind to target analytes. The method used to identify aptamers is Systematic Evolution of Ligands through Exponential Enrichment (SELEX). Target properties can have an impact on aptamer efficiencies. Therefore, characteristics of water-borne microbial targets must be carefully considered during SELEX for optimal aptamer development. Several aptamers have been described for key water-borne pathogens. Here, we provide an exhaustive overview of these aptamers and discuss important microbial aspects to consider when developing such aptamers.

Encapsulation of Russian Olive Water Kefir as an Innovative Functional Drink with High Antioxidant Activity.

Processing of Russian olive water kefir (RWK), as a fermented functional drink made with Russian olive juice and water kefir grains with high antioxidant activity, into powder is crucial for improving its stability for the commercialization of this product. For the first time, this study aimed to encapsulate water kefir microorganisms and bioactive compounds in RWK using carrier materials to develop a synbiotic functional powder using spray drying as an encapsulation method. The goal was maximizing antioxidant activity, product yield, and survival rate of water kefir microorganisms in the produced Russian olive water kefir powder. The optimal spray drying conditions were observed to be at an inlet air temperature of 120ºC, 35 % feed flow rate, and 7 % concentration of drying aid. The effects of spray drying conditions on the quality of microcapsules were assessed and modeled, and the validity of the model was verified. Also, the spray-dried powder's physicochemical properties were assessed and showed promising microbial and physicochemical characteristics compared with the freeze-dried powder.

Local Adaptation of Legionella pneumophila within a Hospital Hot Water System Increases Tolerance to Copper.

In large-building water systems, Legionella pneumophila is exposed to common environmental stressors such as copper. The aim of this study was to evaluate the susceptibility to copper of L. pneumophila isolates recovered from various sites: two clinical and seven environmental isolates from hot water system biofilm and water and from cooling tower water. After a 1-week acclimation in simulated drinking water, strains were exposed to various copper concentrations (0.8 to 5 mg/liter) for over 672 h. Complete loss of culturability was observed for three isolates following copper exposure to 5 mg/liter for 672 h. Two sequence type 1427 (ST1427)-like isolates were highly sensitive to copper, while the other two, isolated from biofilm samples, maintained higher culturability. The expression of the copper resistance gene copA evaluated by reverse transcription-quantitative PCR (RT-qPCR) was significantly higher for the biofilm isolates. All four ST1427-like isolates were recovered from the same water system during an outbreak. Whole-genome sequencing results confirmed that the four isolates are very close phylogenetically, differing by only 29 single nucleotide polymorphisms, suggesting in situ adaptation to microenvironmental conditions, possibly due to epigenetic regulation. These results indicate that the immediate environment within a building water distribution system influences the tolerance of L. pneumophila to copper. Increased contact of L. pneumophila biofilm strains with copper piping or copper alloys in the heat exchanger might lead to local adaptation. The phenotypic differences observed between water and biofilm isolates from the hot water system of a health care facility warrants further investigation to assess the relevance of evaluating disinfection performances based on water sampling alone.IMPORTANCELegionella pneumophila is a pathogen indigenous to natural and large building water systems in the bulk and the biofilm phases. The immediate environment within a system can impact the tolerance of L. pneumophila to environmental stressors, including copper. In health care facilities, copper levels in water can vary, depending on water quality, plumbing materials, and age. This study evaluated the impact of the isolation site (water versus biofilm, hot water system versus cooling tower) within building water systems. Closely related strains isolated from a health care facility hot water system exhibited variable tolerance to copper stress, shown by differential expression of copA, with biofilm isolates displaying highest expression and tolerance. Relying on the detection of L. pneumophila in water samples following exposure to environmental stressors such as copper may underestimate the prevalence of L. pneumophila, leading to inappropriate risk management strategies and increasing the risk of exposure for vulnerable patients.

The Tail-Specific Protease Is Important for Legionella pneumophila To Survive Thermal Stress in Water and inside Amoebae.

Legionella pneumophila (Lp) is an inhabitant of natural and human-made water systems, where it replicates within amoebae and ciliates and survives within biofilms. When Lp-contaminated aerosols are breathed in, Lp can enter the lungs and may infect human alveolar macrophages, causing severe pneumonia known as Legionnaires' disease. Lp is often found in hot water distribution systems (HWDS), which are linked to nosocomial outbreaks. Heat treatment is used to disinfect HWDS and reduce the concentration of Lp However, Lp is often able to recolonize these water systems, indicating an efficient heat shock response. Tail-specific proteases (Tsp) are typically periplasmic proteases implicated in degrading aberrant proteins in the periplasm and important for surviving thermal stress. In Lp Philadelphia-1, Tsp is encoded by the lpg0499 gene. In this paper, we show that Tsp is important for surviving thermal stress in water and for optimal infection of amoeba when a shift in temperature occurs during intracellular growth. We also demonstrate that Tsp is expressed in the postexponential phase but repressed in the exponential phase and that the cis-encoded small regulatory RNA Lpr17 shows the opposite expression, suggesting that it represses translation of tsp In addition, our results show that tsp is regulated by CpxR, a major regulator in Lp, in an Lpr17-independent manner. Deletion of CpxR also reduced the ability of Lp to survive heat shock. In conclusion, our study shows that Tsp is likely an important factor for the survival and growth of Lp in water systems.IMPORTANCELp is a major cause of nosocomial and community-acquired pneumonia. Lp is found in water systems, including hot water distribution systems. Heat treatment is a method of disinfection often used to limit the presence of Lp in such systems; however, the benefit is usually short term, as Lp is able to quickly recolonize these systems. Presumably, Lp responds efficiently to thermal stress, but so far, not much is known about the genes involved. In this paper, we show that the Tsp and the two-component system CpxRA are required for resistance to thermal stress when Lp is free in water and when it is inside host cells. Our study identifies critical systems for the survival of Lp in its natural environment under thermal stress.

The small regulatory RNA Lpr10 regulates the expression of RpoS in Legionella pneumophila.

Legionella pneumophila (Lp) is a waterborne bacterium able to infect human alveolar macrophages, causing Legionnaires' disease. Lp can survive for several months in water, while searching for host cells to grow in, such as ciliates and amoeba. In Lp, the sigma factor RpoS is essential for survival in water. A previous transcriptomic study showed that RpoS positively regulates the small regulatory RNA Lpr10. In the present study, deletion of lpr10 results in an increased survival of Lp in water. Microarray analysis and RT-qPCR revealed that Lpr10 negatively regulates the expression of RpoS in the postexponential phase. Electrophoretic mobility shift assay and in-line probing showed that Lpr10 binds to a region upstream of the previously identified transcription start sites (TSS) of rpoS. A third putative transcription start site was identified by primer extension analysis, upstream of the Lpr10 binding site. In addition, nlpD TSS produces a polycistronic mRNA including the downstream gene rpoS, indicating a fourth TSS for rpoS. Our results suggest that the transcripts from the third and fourth TSS are negatively regulated by the Lpr10 sRNA. Therefore, we propose that Lpr10 is involved in a negative regulatory feedback loop to maintain expression of RpoS to an optimal level.

Unravelling the importance of the eukaryotic and bacterial communities and their relationship with Legionella spp. ecology in cooling towers: a complex network.

BACKGROUND: Cooling towers are a major source of large community-associated outbreaks of Legionnaires' disease, a severe pneumonia. This disease is contracted when inhaling aerosols that are contaminated with bacteria from the genus Legionella, most importantly Legionella pneumophila. How cooling towers support the growth of this bacterium is still not well understood. As Legionella species are intracellular parasites of protozoa, it is assumed that protozoan community in cooling towers play an important role in Legionella ecology and outbreaks. However, the exact mechanism of how the eukaryotic community contributes to Legionella ecology is still unclear. Therefore, we used 18S rRNA gene amplicon sequencing to characterize the eukaryotic communities of 18 different cooling towers. The data from the eukaryotic community was then analysed with the bacterial community of the same towers in order to understand how each community could affect Legionella spp. ecology in cooling towers. RESULTS: We identified several microbial groups in the cooling tower ecosystem associated with Legionella spp. that suggest the presence of a microbial loop in these systems. Dissolved organic carbon was shown to be a major factor in shaping the eukaryotic community and may be an important factor for Legionella ecology. Network analysis, based on co-occurrence, revealed that Legionella was correlated with a number of different organisms. Out of these, the bacterial genus Brevundimonas and the ciliate class Oligohymenophorea were shown, through in vitro experiments, to stimulate the growth of L. pneumophila through direct and indirect mechanisms. CONCLUSION: Our results suggest that Legionella ecology depends on the host community, including ciliates and on several groups of organisms that contribute to its survival and growth in the cooling tower ecosystem. These findings further support the idea that some cooling tower microbiomes may promote the survival and growth of Legionella better than others. Video Abstract.

Identification of two aptamers binding to Legionella pneumophila with high affinity and specificity.

Legionella pneumophila (Lp) is a water borne bacterium causing Legionnaires' Disease (LD) in humans. Rapid detection of Lp in water system is essential to reduce the risk of LD outbreaks. The methods currently available require expert skills and are time intensive, thus delaying intervention. In situ detection of Lp by biosensor would allow rapid implementation of control strategies. To this end, a biorecognition element is required. Aptamers are considered promising biorecognition molecules for biosensing. Aptamers are short oligonucleotide sequence folding into a specific structure and are able to bind to specific molecules. Currently, no aptamer and thus no aptamer-based technology exists for the detection of Lp. In this study, Systemic Evolution of Ligands through EXponential enrichment (SELEX) was used to identify aptamers binding specifically to Lp. Ten rounds of positive selection and two rounds of counter-selection against two Pseudomonas species were performed. Two aptamers binding strongly to Lp were identified with KD of 116 and 135 nM. Binding specificity of these two aptamers to Lp was confirmed by flow cytometry and fluorescence microscopy. Therefore, these two aptamers are promising biorecognition molecules for the detection of Lp in water systems.

Impact of temperature on Legionella pneumophila, its protozoan host cells, and the microbial diversity of the biofilm community of a pilot cooling tower.

Legionella pneumophila is a waterborne bacterium known for causing Legionnaires' Disease, a severe pneumonia. Cooling towers are a major source of outbreaks, since they provide ideal conditions for L. pneumophila growth and produce aerosols. In such systems, L. pneumophila typically grow inside protozoan hosts. Several abiotic factors such as water temperature, pipe material and disinfection regime affect the colonization of cooling towers by L. pneumophila. The local physical and biological factors promoting the growth of L. pneumophila in water systems and its spatial distribution are not well understood. Therefore, we built a lab-scale cooling tower to study the dynamics of L. pneumophila colonization in relationship to the resident microbiota and spatial distribution. The pilot was filled with water from an operating cooling tower harboring low levels of L. pneumophila. It was seeded with Vermamoeba vermiformis, a natural host of L. pneumophila, and then inoculated with L. pneumophila. After 92 days of operation, the pilot was disassembled, the water was collected, and biofilm was extracted from the pipes. The microbiome was studied using 16S rRNA and 18S rRNA genes amplicon sequencing. The communities of the water and of the biofilm were highly dissimilar. The relative abundance of Legionella in water samples reached up to 11% whereas abundance in the biofilm was extremely low (≤0.5%). In contrast, the host cells were mainly present in the biofilm. This suggests that L. pneumophila grows in host cells associated with biofilm and is then released back into the water following host cell lysis. In addition, water temperature shaped the bacterial and eukaryotic community of the biofilm, indicating that different parts of the systems may have different effects on Legionella growth.

Legionella quinlivanii strain isolated from a human: A case report and whole genome sequencing analysis.

We describe a strain of Legionella quinlivanii isolated from a bronchoalveolar lavage specimen from an 83-year-old patient in the province of Québec. Identification was done using 16S rRNA sequencing. The strain could replicate efficiently in human THP-1 macrophages and maintained a low level of cytotoxicity. Upon analyzing the whole genome sequencing data, the icm/dot secretion system was present, but the strain lacked some effector genes known to express proteins toxic to cells. The pathogenicity of this Legionella species should be investigated further.

Les auteurs décrivent une souche de Legionella quinlivanii isolée dans le prélèvement de lavage bronchoalvéolaire d'une patiente de 83 ans de la province de Québec. Ils ont identifié la souche par séquençage de l'ARN ribosomal 16S. Cette souche, qui pouvait se répliquer en toute efficacité dans les macrophages humains THP-1, maintenait une faible cytotoxicité. L'analyse des données de séquençage complet du génome de la souche a révélé la présence du système de sécrétion icm/dot, mais l'absence de certains gènes effecteurs connus pour exprimer les protéines cytotoxiques. Il faudra étudier plus en profondeur la pathogénicité de cette espèce de Legionella.

Presence of Legionella spp. in cooling towers: the role of microbial diversity, Pseudomonas, and continuous chlorine application.

Legionnaires' disease (LD) is a severe pneumonia caused by several species of the genus Legionella, most frequently by Legionella pneumophila. Cooling towers are the most common source for large community-associated outbreaks. Colonization, survival, and proliferation of L. pneumophila in cooling towers are necessary for outbreaks to occur. These steps are affected by the chemical and physical parameters of the cooling tower environment. We hypothesize that the bacterial community residing in the cooling tower could also affect the presence of L. pneumophila. A 16S rRNA gene targeted amplicon sequencing approach was used to study the bacterial community of cooling towers and its relationship with the Legionella spp. and L. pneumophila communities. The results indicated that the water source shaped the bacterial community of cooling towers. Several taxa were enriched and positively correlated with Legionella spp. and L. pneumophila. In contrast, Pseudomonas showed a strong negative correlation with Legionella spp. and several other genera. Most importantly, continuous chlorine application reduced microbial diversity and promoted the presence of Pseudomonas creating a non-permissive environment for Legionella spp. This suggests that disinfection strategies as well as the resident microbial population influences the ability of Legionella spp. to colonize cooling towers.

Legionella pneumophila levels and sequence-type distribution in hospital hot water samples from faucets to connecting pipes.

Recent studies have reported increased levels of Legionella pneumophila (Lp) at points of use compared to levels in primary and secondary components of hot water systems, suggesting possible selection by environmental conditions. In this study, concentrations of Lp in a hospital hot water system were evaluated by profile sampling, collecting successive water samples to determine the prevalence at the faucet (distal) and upstream piping before and after a system intervention to increase temperature. Lp strain diversity was compared between different points of use and different areas of the hot water system (i.e., tap, intermediate piping and main upflow piping). In total, 47 isolates were recovered from 32 positive hot water samples collected from designated taps, showers and recirculation loops; these isolates were subsequently analyzed by sequence-based typing (SBT). Lp levels were comparable between first draw (500 mL) and flushed (2 and 5 min) samples, whereas a decrease was observed in the amount of culturable cells (1 log). Two sequence types (STs) were identified throughout the system. ST378 (sg4/10) was present in 91% of samples, while ST154-like (sg1) was present in 41%; both STs were simultaneously recovered in 34% of samples. Isolated STs displayed comparable tolerance to copper (0.8-5 mg/L) and temperature (55 °C, 1 h) exposure. The ability to replicate within THP1 cells and Acanthamoeba castellanii was similar between the two STs and a comparative environmental outbreak strain. The low Lp diversity and the detection of both Lp sequence types in repeated subsequent samples collected from positive faucets in a hospital wing suggest a minimal impact of the distal conditions on strain selection for the sampled points, as well as a possible adaptation to stressors present in the system, leading to the predominance of a few strains.

Quantification of Viable but Non-Culturable Cells of Legionella pneumophila.

Legionella pneumophila, among other bacteria, may enter a viable but non-culturable state as a means for survival in stressful conditions. Bacterial cells in the viable but non-culturable state cannot grow on standard medium; however, they continue to exhibit characteristics that are associated with live cells, such as respiration, transcription, and cell wall integrity. The present paper outlines a detailed protocol for the detection of viable but non-culturable L. pneumophila cells via Syto® 9 and propidium iodide staining coupled with flow cytometry.

The Virulence Effect of CpxRA in Citrobacter rodentium Is Independent of the Auxiliary Proteins NlpE and CpxP.

Citrobacter rodentium is a murine pathogen used to model the intestinal infection caused by Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC and EHEC), two diarrheal pathogens responsible for morbidity and mortality in developing and developed countries, respectively. During infection, these bacteria must sense and adapt to the gut environment of the host. In order to adapt to changing environmental cues and modulate expression of specific genes, bacteria can use two-component signal transduction systems (TCS). We have shown that the deletion of the Cpx TCS in C. rodentium leads to a marked attenuation in virulence in C3H/HeJ mice. In E. coli, the Cpx TCS is reportedly activated in response to signals from the outer-membrane lipoprotein NlpE. We therefore investigated the role of NlpE in C. rodentium virulence. We also assessed the role of the reported negative regulator of CpxRA, CpxP. We found that as opposed to the ΔcpxRA strain, neither the ΔnlpE, ΔcpxP nor the ΔnlpEΔcpxP strains were significantly attenuated, and had similar in vivo localization to wild-type C. rodentium. The in vitro adherence of the Cpx auxiliary protein mutants, ΔnlpE, ΔcpxP, ΔnlpEΔcpxP, was comparable to wild-type C. rodentium, whereas the ΔcpxRA strain showed significantly decreased adherence. To further elucidate the mechanisms behind the contrasting virulence phenotypes, we performed microarrays in order to define the regulon of the Cpx TCS. We detected 393 genes differentially regulated in the ΔcpxRA strain. The gene expression profile of the ΔnlpE strain is strikingly different than the profile of ΔcpxRA with regards to the genes activated by CpxRA. Further, there is no clear inverse correlation in the expression pattern of the ΔcpxP strain in comparison to ΔcpxRA. Taken together, these data suggest that in these conditions, CpxRA activates gene expression in a largely NlpE- and CpxP-independent manner. Compared to wildtype, 161 genes were downregulated in the ΔcpxRA strain, while being upregulated or unchanged in the Cpx auxiliary protein deletion strains. This group of genes, which we hypothesize may contribute to the loss of virulence of ΔcpxRA, includes T6SS components, ompF, the regulator for colanic acid synthesis, and several genes involved in maltose metabolism.