Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Conventional antimicrobials trigger an SOS response in EHEC that promotes the release of the potent Shiga toxin that is responsible for much of the morbidity and mortality associated with EHEC infection. Cattle are a natural reservoir of EHEC, and approximately 75% of EHEC outbreaks are linked to the consumption of contaminated bovine-derived products. This review will discuss how EHEC causes disease in humans but is asymptomatic in adult ruminants. It will also analyze factors utilized by EHEC as it travels through the bovine gastrointestinal (GI) tract that allow for its survival through the acidic environment of the distal stomachs, and for its ultimate colonization in the recto-anal junction (RAJ). Understanding the factors crucial for EHEC survival and colonization in cattle will aid in the development of alternative strategies to prevent EHEC shedding into the environment and consequent human infection.
Gastrointestinal (GI) bacteria sense diverse environmental signals as cues for differential gene regulation and niche adaptation. Pathogens such as enterohemorrhagic Escherichia coli (EHEC), which causes bloody diarrhea, use these signals for the temporal and energy-efficient regulation of their virulence factors. One of the main virulence strategies employed by EHEC is the formation of attaching and effacing (AE) lesions on enterocytes. Most of the genes necessary for the formation of these lesions are grouped within a pathogenicity island, the locus of enterocyte effacement (LEE), whose expression requires the LEE-encoded regulator Ler. Here we show that growth of EHEC in glycolytic environments inhibits the expression of ler and consequently all other LEE genes. Conversely, growth within a gluconeogenic environment activates expression of these genes. This sugar-dependent regulation is achieved through two transcription factors: KdpE and Cra. Both Cra and KdpE directly bind to the ler promoter, and Cra’s affinity to this promoter is catabolite dependent. Moreover, we show that the Cra and KdpE proteins interact in vitro and that KdpE’s ability to bind DNA is enhanced by the presence of Cra. Cra is important for AE lesion formation, and KdpE contributes to this Cra-dependent regulation. The deletion of cra and kdpE resulted in the ablation of AE lesions. One of the many challenges that bacteria face within the GI tract is to successfully compete for carbon sources. Linking carbon metabolism to the precise coordination of virulence expression is a key step in the adaptation of pathogens to the GI environment.
For unsuspecting bacteria, the difference between life and death depends upon efficient and specific responses to various stressors. Facing a much larger world, microbes are invariably challenged with ever-changing environments where temperature, pH, chemicals, and nutrients are in a constant state of flux. Only those that are able to rapidly reprogram themselves and express subsets of genes needed to overcome the stress will survive and outcompete neighboring microbes. Recently, low shear stress, emulating microgravity (MG) experienced in space, has been characterized in a number of microorganisms including fungi and prokaryotes ranging from harmless surrogate organisms to bona fide pathogens. Interestingly, MG appears to induce a plethora of effects ranging from enhanced pathogenicity in several Gram-negative enterics to enhanced biofilm formation. Furthermore, MG-exposed bacteria appeared better able to handle subsequent stressors including: osmolarity, pH, temperature, and antimicrobial challenge while yeast exhibited aberrant budding post-MG-exposure. This review will focus on MG-induced alterations of virulence in various microbes with the emphasis placed on bacteria.
Bacteria engage in chemical signaling, termed quorum sensing (QS), to mediate intercellular communication, mimicking multicellular organisms. The LuxR family of QS transcription factors regulates gene expression, coordinating population behavior by sensing endogenous acyl homoserine lactones (AHLs). However, some bacteria (such as Escherichia coli) do not produce AHLs. These LuxR orphans sense exogenous AHLs but also regulate transcription in the absence of AHLs. Importantly, this AHL-independent regulatory mechanism is still largely unknown. Here we present several structures of one such orphan LuxR-type protein, SdiA, from enterohemorrhagic E. coli (EHEC), in the presence and absence of AHL. SdiA is actually not in an apo state without AHL but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life and serves as an energy source, signaling molecule, and substrate for membrane biogenesis. While exogenous AHL renders to SdiA higher stability and DNA binding affinity, OCL may function as a chemical chaperone placeholder that stabilizes SdiA, allowing for basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides crucial mechanistic insights into the ligand regulation of AHL-dependent and -independent function of LuxR-type proteins. Importantly, in addition to its contribution to basic science, this work has implications for public health, inasmuch as the SdiA signaling system aids the deadly human pathogen EHEC to adapt to a commensal lifestyle in the gastrointestinal (GI) tract of cattle, its main reservoir. These studies open exciting and novel avenues to control shedding of this human pathogen in the environment.
Several viruses encounter various bacterial species within the host and in the environment. Despite these close encounters, the effects of bacteria on picornaviruses are not completely understood. Previous work determined that poliovirus (PV), an enteric virus, has enhanced virion stability when exposed to bacteria or bacterial surface polysaccharides such as lipopolysaccharide. Virion stabilization by bacteria may be important for interhost transmission, since a mutant PV with reduced bacterial binding had a fecal-oral transmission defect in mice. Therefore, we investigated whether bacteria broadly enhance stability of picornaviruses from three different genera: Enterovirus (PV and coxsackievirus B3 [CVB3]), Kobuvirus (Aichi virus), and Cardiovirus (mengovirus). Furthermore, to delineate strain-specific effects, we examined two strains of CVB3 and a PV mutant with enhanced thermal stability. We determined that specific bacterial strains enhance thermal stability of PV and CVB3, while mengovirus and Aichi virus are stable at high temperatures in the absence of bacteria. Additionally, we determined that bacteria or lipopolysaccharide can stabilize PV, CVB3, Aichi virus, and mengovirus during exposure to bleach. These effects are likely mediated through direct interactions with bacteria, since viruses bound to bacteria in a pulldown assay. Overall, this work reveals shared and distinct effects of bacteria on a panel of picornaviruses. IMPORTANCE Recent studies have shown that bacteria promote infection and stabilization of poliovirus particles, but the breadth of these effects on other members of the Picornaviridae family is unknown. Here, we compared the effects of bacteria on four distinct members of the Picornaviridae family. We found that bacteria reduced inactivation of all of the viruses during bleach treatment, but not all viral strains were stabilized by bacteria during heat treatment. Overall, our data provide insight into how bacteria play differential roles in picornavirus stability.
Enteric viruses, including poliovirus, are spread by the fecal-oral route. In order to persist and transmit to a new host, enteric virus particles must remain stable once they are in the environment. Environmental stressors such as heat and disinfectants can inactivate virus particles and prevent viral transmission. It has been previously demonstrated that bacteria or bacterial surface glycans can enhance poliovirus virion stability and limit inactivation from heat or bleach. While investigating the mechanisms underlying bacterially enhanced virion thermal stability, we identified and characterized a poliovirus (PV) mutant with increased resistance to heat inactivation. The M132V mutant harbors a single amino acid change in the VP1 capsid coding that is sufficient to confer heat resistance but not bleach resistance. Although the M132V virus was stable in the absence of bacteria or feces at most temperatures, M132V virus was stabilized by feces at very high temperatures. M132V PV had reduced specific infectivity and RNA uncoating compared with those of wild-type (WT) PV, but viral yields in HeLa cells were similar. In orally inoculated mice, M132V had a slight fitness cost since fecal titers were lower and 12.5% of fecal viruses reverted to the WT. Overall, this work sheds light on factors that influence virion stability and fitness. IMPORTANCE Viruses spread by the fecal-oral route need to maintain viability in the environment to ensure transmission. Previous work indicated that bacteria and bacterial surface polysaccharides can stabilize viral particles and enhance transmission. To explore factors that influence viral particle stability, we isolated a mutant poliovirus that is heat resistant. This mutant virus does not require feces for stability at most temperatures but can be stabilized by feces at very high temperatures. Even though the mutant virus is heat resistant, it is susceptible to inactivation by treatment with bleach. This work provides insight into how viral particles maintain infectivity in the environment.
bEnterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis and life-threatening complications. The main reservoirs for EHEC are healthy ruminants. We reported that SdiA senses acyl homoserine lactones (AHLs) in the bovine rumen to activate expression of the glutamate acid resistance (gad) genes priming EHEC's acid resistance before they pass into the acidic abomasum. Conversely, SdiA represses expression of the locus of enterocyte effacement (LEE) genes, whose expression is not required for bacterial survival in the rumen but is necessary for efficient colonization at the rectoanal junction (RAJ) mucosa. Our previous studies show that SdiA-dependent regulation was necessary for efficient EHEC colonization of cattle fed a grain diet. Here, we compared the SdiA role in EHEC colonization of cattle fed a forage hay diet. We detected AHLs in the rumen of cattle fed a hay diet, and these AHLs activated gad gene expression in an SdiA-dependent manner. The rumen fluid and fecal samples from hay-fed cattle were near neutrality, while the same digesta samples from grain-fed animals were acidic. Cattle fed either grain or hay and challenged with EHEC orally carried the bacteria similarly. EHEC was cleared from the rumen within days and from the RAJ mucosa after approximately one month. In competition trials, where animals were challenged with both wild-type and SdiA deletion mutant bacteria, diet did not affect the outcome that the wild-type strain was better able to persist and colonize. However, the wild-type strain had a greater advantage over the SdiA deletion mutant at the RAJ mucosa among cattle fed the grain diet.
The bacterial predator Bdellovibrio bacteriovorus is evolved to attack and kill other bacteria, including the human intestinal pathogen Vibrio cholerae. Although B. bacteriovorus exhibit a broad prey range, little is known about the genetic determinants of prey resistance and sensitivity. Here we perform a genetic screen on V. cholerae and identify five pathways contributing to predation susceptibility. We find that the essential virulence regulators ToxR/S increase susceptibility to predation, as mutants of these genes are more resistant to predation. We observe by flow cytometry that lipopolysaccharide is a critical defense, as mutants lacking O-antigen are rapidly attacked by predatory B. bacteriovorus. Using polymer solutions to alter media viscosity, we find that when B. bacteriovorus attacks motile V. cholerae, increased drag forces slow its ability to prey. These results provide insights into key prey resistance mechanisms, and may be useful in the application of B. bacteriovorus in treating infections.
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