Brian M. M. Ahmer

Cell‐to‐cell signalling in Escherichia coli and Salmonella enterica

Cell‐to‐cell signalling in prokaryotes that leads to co‐ordinated behaviour has been termed quorum sensing. This type of signalling can have profound impacts on microbial community structure and host–microbe interactions. The Gram‐negative quorum‐sensing systems were first discovered and extensively characterized in the marine Vibrios. Some components of the Vibrio systems are present in the classical genetic model organisms Escherichia coli and Salmonella enterica. Both organisms encode a signal receptor of the LuxR family, SdiA, but not a corresponding signal‐generating enzyme. Instead, SdiA of Salmonella detects and responds to signals generated only by other microbial species. Conversely, E. coli and Salmonella encode the signal‐generating component of a second system (a LuxS homologue that generates AI‐2), but the sensory apparatus for AI‐2 differs substantially from the Vibrio system. The only genes currently known to be regulated by AI‐2 in Salmonella encode an active uptake and modification system for AI‐2. Therefore, it is not yet clear whether Salmonella uses AI‐2 as a signal molecule or whether AI‐2 has some other function. In E. coli, the functions of both SdiA and AI‐2 are unclear due to pleiotropy. Genetic strategies to identify novel signalling systems have been performed with E. coli and Providencia stuartii. Several putative signalling systems have been identified, one that uses indole as a signal and another that releases what appears to be a peptide. The latter system has homologues in E. coli and Salmonella, as well as other bacteria, plants and animals. In fact, the protease components from Providencia and Drosophila are functionally interchangeable.

SdiA of Salmonella enterica Is a LuxR Homolog That Detects Mixed Microbial Communities

Proteins of the LuxR family detect the presence of N-acylhomoserine lactones (AHLs) and regulate transcription accordingly. When AHLs are synthesized by the same species that detects them, the system allows a bacterium to measure the population density of its own species, a phenomenon known as quorum sensing. The sdiA genes of Escherichia coli and Salmonella enterica serovar Typhimurium are predicted to encode LuxR homologs. However, these species do not appear to synthesize AHLs or any other molecule detected by SdiA. It has previously been demonstrated that overexpression of sdiA results in the activation of the ftsQAZ locus in E. coli and four other loci in Salmonella serovar Typhimurium. Here we report that transcriptional fusions to these five loci fall into two classes. The first class requires overexpression of sdiA for activation. The second class responds to sdiA expressed from its natural position in the chromosome if the appropriate AHLs are added to the culture. The only member of the second class is a series of Prck-luxCDABE fusions in Salmonella serovar Typhimurium. SdiA responds with highest sensitivity to AHLs that have a keto modification at the third carbon and an acyl chain length of 6 or 8 (half-maximal response between 1 and 5 nM). Growth of Salmonella in proximity to species known to synthesize these AHLs results in sdiA-dependent activation of the Prck-luxCDABE fusions. SdiA appears to be the first AHL receptor discovered that detects signals emanating exclusively from other species.

Regulation of Enteric Endophytic Bacterial Colonization by Plant Defenses

Bacterial endophytes reside within the interior of plants without causing disease or forming symbiotic structures. Some endophytes, such as Klebsiella pneumoniae 342 (Kp342), enhance plant growth and nutrition. Others, such as Salmonella enterica serovar Typhimurium (S. typhimurium), are human pathogens that contaminate raw produce. Several lines of evidence are presented here to support the hypothesis that plant defense response pathways regulate colonization by endophytic bacteria. An ethylene-insensitive mutant of Medicago truncatula is hypercolonized by Kp342 compared to the parent genotype. Addition of ethylene, a signal molecule for induced systemic resistance in plants, decreased endophytic colonization in Medicago spp. This ethylene-mediated inhibition of endophytic colonization was reversed by addition of the ethylene action inhibitor, 1-methylcyclopropene. Colonization of Medicago spp. by S. typhimurium also was affected by exogenous ethylene. Mutants lacking flagella or a component of the type III secretion system of Salmonella pathogenicity island 1 (TTSS-SPI1) colonize the interior of Medicago spp. in higher numbers than the wild type. Arabidopsis defense response-related genotypes indicated that only salicylic acid (SA)-independent defense responses contribute to restricting colonization by Kp342. In contrast, colonization by S. typhimurium is affected by both SA-dependent and -independent responses. S. typhimurium mutants further delineated these responses, suggesting that both flagella and TTSS-SPI1 effectors can be recognized. Flagella act primarily through SA-independent responses (compromising SA accumulation still affected colonization in the absence of flagella). Removal of a TTSS-SPI1 effector resulted in hypercolonization regardless of whether the genotype was affected in either SA-dependent or SA-independent responses. Consistent with these results, S. typhimurium activates the promoter of PR1, a SA-dependent pathogenesis-related gene, while S. typhimurium mutants lacking the TTSS-SPI1 failed to activate this promoter. These observations suggest approaches to reduce contamination of raw produce by human enteric pathogens and to increase the number of growth-promoting bacteria in plants.

Kinetics and strain specificity of rhizosphere and endophytic colonization by enteric bacteria on seedlings of Medicago sativa and Medicago truncatula.

ABSTRACT The presence of human-pathogenic, enteric bacteria on the surface and in the interior of raw produce is a significant health concern. Several aspects of the biology of the interaction between these bacteria and alfalfa (Medicago sativa) seedlings are addressed here. A collection of enteric bacteria associated with alfalfa sprout contaminations, along with Escherichia coli K-12, Salmonella enterica serotype Typhimurium strain ATCC 14028, and an endophyte of maize, Klebsiella pneumoniae 342, were labeled with green fluorescent protein, and their abilities to colonize the rhizosphere and the interior of the plant were compared. These strains differed widely in their endophytic colonization abilities, with K. pneumoniae 342 and E. coli K-12 being the best and worst colonizers, respectively. The abilities of the pathogens were between those of K. pneumoniae 342 and E. coli K-12. All Salmonella bacteria colonized the interiors of the seedlings in high numbers with an inoculum of 102 CFU, although infection characteristics were different for each strain. For most strains, a strong correlation between endophytic colonization and rhizosphere colonization was observed. These results show significant strain specificity for plant entry by these strains. Significant colonization of lateral root cracks was observed, suggesting that this may be the site of entry into the plant for these bacteria. At low inoculum levels, a symbiosis mutant of Medicago truncatula, dmi1, was colonized in higher numbers on the rhizosphere and in the interior by a Salmonella endophyte than was the wild-type host. Endophytic entry of M. truncatula appears to occur by a mechanism independent of the symbiotic infections by Sinorhizobium meliloti or mycorrhizal fungi.

Regulation of Bacterial Virulence by Csr (Rsm) Systems

SUMMARY Most bacterial pathogens have the remarkable ability to flourish in the external environment and in specialized host niches. This ability requires their metabolism, physiology, and virulence factors to be responsive to changes in their surroundings. It is no surprise that the underlying genetic circuitry that supports this adaptability is multilayered and exceedingly complex. Studies over the past 2 decades have established that the CsrA/RsmA proteins, global regulators of posttranscriptional gene expression, play important roles in the expression of virulence factors of numerous proteobacterial pathogens. To accomplish these tasks, CsrA binds to the 5′ untranslated and/or early coding regions of mRNAs and alters translation, mRNA turnover, and/or transcript elongation. CsrA activity is regulated by noncoding small RNAs (sRNAs) that contain multiple CsrA binding sites, which permit them to sequester multiple CsrA homodimers away from mRNA targets. Environmental cues sensed by two-component signal transduction systems and other regulatory factors govern the expression of the CsrA-binding sRNAs and, ultimately, the effects of CsrA on secretion systems, surface molecules and biofilm formation, quorum sensing, motility, pigmentation, siderophore production, and phagocytic avoidance. This review presents the workings of the Csr system, the paradigm shift that it generated for understanding posttranscriptional regulation, and its roles in virulence networks of animal and plant pathogens.

SdiA, an N-acylhomoserine lactone receptor, becomes active during the transit of Salmonella enterica through the gastrointestinal tract of turtles

Background LuxR-type transcription factors are typically used by bacteria to determine the population density of their own species by detecting N-acylhomoserine lactones (AHLs). However, while Escherichia and Salmonella encode a LuxR-type AHL receptor, SdiA, they cannot synthesize AHLs. In vitro, it is known that SdiA can detect AHLs produced by other bacterial species. Methodology/Principal Findings In this report, we tested the hypothesis that SdiA detects the AHL-production of other bacterial species within the animal host. SdiA did not detect AHLs during the transit of Salmonella through the gastrointestinal tract of a guinea pig, a rabbit, a cow, 5 mice, 6 pigs, or 12 chickens. However, SdiA was activated during the transit of Salmonella through turtles. All turtles examined were colonized by the AHL-producing species Aeromonas hydrophila. Conclusions/Significance We conclude that the normal gastrointestinal microbiota of most animal species do not produce AHLs of the correct type, in an appropriate location, or in sufficient quantities to activate SdiA. However, the results obtained with turtles represent the first demonstration of SdiA activity in animals.

E. coli K-12 and EHEC Genes Regulated by SdiA

Background Escherichia and Salmonella encode SdiA, a transcription factor of the LuxR family that regulates genes in response to N-acyl homoserine lactones (AHLs) produced by other species of bacteria. E. coli genes that change expression in the presence of plasmid-encoded sdiA have been identified by several labs. However, many of these genes were identified by overexpressing sdiA on a plasmid and have not been tested for a response to sdiA produced from its natural position in the chromosome or for a response to AHL. Methodology/Principal Findings We determined that two important loci reported to respond to plasmid-based sdiA, ftsQAZ and acrAB, do not respond to sdiA expressed from its natural position in the chromosome or to AHLs. To identify genes that are regulated by chromosomal sdiA and/or AHLs, we screened 10,000 random transposon-based luciferase fusions in E. coli K-12 and a further 10,000 in E. coli O157:H7 for a response to AHL and then tested these genes for sdiA-dependence. We found that genes encoding the glutamate-dependent acid resistance system are up-regulated, and fliE is down-regulated, by sdiA. Gene regulation by sdiA of E. coli is only partially dependent upon AHL. Conclusions/Significance The genes of E. coli that respond to plasmid-based expression of sdiA are largely different than those that respond to chromosomal sdiA and/or AHL. This has significant implications for determining the true function of AHL detection by E. coli.

Salmonella SdiA Recognizes N-acyl Homoserine Lactone Signals from Pectobacterium carotovorum in Vitro, but Not in a Bacterial Soft Rot

Genomes of Salmonella enterica isolates, including those linked to outbreaks of produce-associated gastroenteritis, contain sdiA, which encodes a receptor of N-acyl homoserine lactones (AHL). AHL are the quorum-sensing signals used by bacteria to coordinately regulate gene expression within -their populations. Because S. enterica does not produce its own AHL, SdiA is hypothesized to function in the interspecies cross-talk with AHL-producing bacteria. Under laboratory conditions, S. enterica responded to AHL from phytobacteria by upregulating expression of srgE. AHL-dependent expression of srgE required a functional sdiA. Essentially, no sdiA-dependent resolution of the srgE recombinase-based (RIVET) reporter was observed inside a soft rot formed on a tomato by an AHL-producing strain of Pectobacterium carotovorum. The results of the control experiments suggest that sdiA is not expressed inside tomato, pepper, green onion, or carrot affected by the soft rot, and the lack of sdiA expression in planta prevents Salmonella spp. from responding to AHL. Despite its inability to detect and respond to AHL during colonization of soft rots, S. enterica reached higher final cell numbers inside a tomato soft rot compared with its growth in intact tomato fruit. The synergistic effect was the strongest under the conditions that are typical for the Florida fall/winter production season.

The Intestinal Microbiota Are Necessary for Stressor-Induced Enhancement of Splenic Macrophage Microbicidal Activity

The indigenous microbiota impact mucosal, as well as systemic, immune responses, but whether the microbiota are involved in stressor-induced immunomodulation has not been thoroughly tested. A well characterized murine stressor, called social disruption (SDR), was used to study whether the microbiota are involved in stressor-induced enhancement of macrophage reactivity. Exposure to the SDR Stressor enhanced the ability of splenic macrophages to produce microbicidal mediators (e.g., inducible nitric oxide synthase (iNOS), superoxide anion, and peroxynitrite) and to kill target Escherichia coli. Exposure to the SDR Stressor also increased cytokine production by LPS-stimulated splenic macrophages. These effects, however, were impacted by the microbiota. Microbicidal activity and cytokine mRNA in splenic macrophages from Swiss Webster germfree mice that lack any commensal microbiota were not enhanced by exposure to the SDR Stressor. However, when germfree mice were conventionalized by colonizing them with microbiota from CD1 conventional donor mice, exposure to the SDR Stressor again increased microbicidal activity and cytokine mRNA. In follow-up experiments, immunocompetent conventional CD1 mice were treated with a cocktail of antibiotics to disrupt the intestinal microbiota. While exposure to the SDR Stressor-enhanced splenic macrophage microbicidal activity and cytokine production in vehicle-treated mice, treatment with antibiotics attenuated the SDR Stressor-induced increases in splenic macrophage reactivity. Treatment with antibiotics also prevented the stressor-induced increase in circulating levels of bacterial peptidoglycan, suggesting that translocation of microbiota-derived peptidoglycan into the body primes the innate immune system for enhanced activity. This study demonstrates that the microbiota play a crucial role in stressor-induced immunoenhancement.

Interaction of Salmonella spp. with the Intestinal Microbiota

Salmonella spp. are major cause of human morbidity and mortality worldwide. Upon entry into the human host, Salmonella spp. must overcome the resistance to colonization mediated by the gut microbiota and the innate immune system. They successfully accomplish this by inducing inflammation and mechanisms of innate immune defense. Many models have been developed to study Salmonella spp. interaction with the microbiota that have helped to identify factors necessary to overcome colonization resistance and to mediate disease. Here we review the current state of studies into this important pathogen/microbiota/host interaction in the mammalian gastrointestinal tract.