Small Regulatory RNA and Legionella pneumophila

Legionella pneumophila is a gram-negative bacterial species that is ubiquitous in almost any aqueous environment. It is the agent of Legionnaires’ disease, an acute and often under-reported form of pneumonia. In mammals, L. pneumophila replicates inside macrophages within a modified vacuole. Many protein regulators have been identified that control virulence-related properties, including RpoS, LetA/LetS, and PmrA/PmrB. In the past few years, the importance of regulation of virulence factors by small regulatory RNA (sRNAs) has been increasingly appreciated. This is also the case in L. pneumophila where three sRNAs (RsmY, RsmZ, and 6S RNA) were recently shown to be important determinants of virulence regulation and 79 actively transcribed sRNAs were identified. In this review we describe current knowledge about sRNAs and their regulatory properties and how this relates to the known regulatory systems of L. pneumophila. We also provide a model for sRNA-mediated control of gene expression that serves as a framework for understanding the regulation of virulence-related properties of L. pneumophila

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

ABSTRACT: 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

Energy conservation and the promotion of Legionella pneumophila growth: The probable role of heat exchangers in a nosocomial outbreak

OBJECTIVE To determine the source of a Legionella pneumophila serogroup 5 nosocomial outbreak and the role of the heat exchanger installed on the hot water system within the previous year. SETTING A 400-bed tertiary care university hospital in Sherbrooke, Canada. METHODS Hot water samples were collected and cultured for L. pneumophila from 25 taps (baths and sinks) within wing A and 9 taps in wing B. Biofilm (5) and 2 L water samples (3) were collected within the heat exchangers for L. pneumophila culture and detection of protists. Sequence-based typing was performed on strain DNA extracts and pulsed-field gel electrophoresis patterns were analyzed. RESULTS Following 2 cases of hospital-acquired legionellosis, the hot water system investigation revealed a large proportion of L. pneumophila serogroup 5 positive taps (22/25 in wing A and 5/9 in wing B). High positivity was also detected in the heat exchanger of wing A in water samples (3/3) and swabs from the heat exchanger (4/5). The outbreak genotyping investigation identified the hot water system as the source of infections. Genotyping results revealed that all isolated environmental strains harbored the same related pulsed-field gel electrophoresis pattern and sequence-based type. CONCLUSIONS Two cases of hospital-acquired legionellosis occurred in the year following the installation of a heat exchanger to preheat hospital hot water. No cases were reported previously, although the same L. pneumophila strain was isolated from the hot water system in 1995. The heat exchanger promoted L. pneumophila growth and may have contributed to confirmed clinical cases. Infect. Control Hosp. Epidemiol. 2016;1475-1480

Selection of Salmonella enterica Serovar Typhi Genes Involved during Interaction with Human Macrophages by Screening of a Transposon Mutant Library

The human-adapted Salmonella enterica serovar Typhi (S. Typhi) causes a systemic infection known as typhoid fever. This disease relies on the ability of the bacterium to survive within macrophages. In order to identify genes involved during interaction with macrophages, a pool of approximately 105 transposon mutants of S. Typhi was subjected to three serial passages of 24 hours through human macrophages. Mutants recovered from infected macrophages (output) were compared to the initial pool (input) and those significantly underrepresented resulted in the identification of 130 genes encoding for cell membrane components, fimbriae, flagella, regulatory processes, pathogenesis, and many genes of unknown function. Defined deletions in 28 genes or gene clusters were created and mutants were evaluated in competitive and individual infection assays for uptake and intracellular survival during interaction with human macrophages. Overall, 26 mutants had defects in the competitive assay and 14 mutants had defects in the individual assay. Twelve mutants had defects in both assays, including acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, SPI-4, STY1867-68, and STY2346. The complementation of several mutants by expression of plasmid-borne wild-type genes or gene clusters reversed defects, confirming that the phenotypic impairments within macrophages were gene-specific. In this study, 35 novel phenotypes of either uptake or intracellular survival in macrophages were associated with Salmonella genes. Moreover, these results reveal several genes encoding molecular mechanisms not previously known to be involved in systemic infection by human-adapted typhoidal Salmonella that will need to be elucidated

Role of the LuxR family transcriptional regulator Lpg2524 in the survival of Legionella pneumophila in water

The water-borne Gram-negative bacterium Legionella pneumophila (Lp) is the causative agent of Legionnairesâ disease. Lp is typically transmitted to human from water systems, where it grows inside amoebae. Survival of Lp in water is central to its transmission to humans. A transcriptomic study previously identified many genes induced by Lp in water. One of such gene, lpg2524, encodes a putative LuxR family transcriptional regulator. It was hypothesized that this gene could be involved in the survival of Lp in water. Deletion of lpg2524 does not affect the growth of Lp in rich medium, in the amoeba Acanthamoeba castellanii or in human macrophage-like THP-1 cells, showing that Lpg2524 is not required for growth in vitro and in vivo. Nevertheless, deletion of lpg2524 results in a faster CFU reduction in an artificial freshwater medium, Fraquil, indicating that Lpg2524 is important for Lp to survive in water. Over-expression of Lpg2524 also results in a survival defect, suggesting that a precise level of this transcriptional regulator is essential for its function. However, our result shows that Lpg2524 is dispensable for survival in water when Lp is at a high cell density (10The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Short-Term and Long-Term Survival and Virulence of Legionella pneumophila in the Defined Freshwater Medium Fraquil.

Legionella pneumophila (Lp) is the etiological agent responsible for Legionnaires' disease, a potentially fatal pulmonary infection. Lp lives and multiplies inside protozoa in a variety of natural and man-made water systems prior to human infection. Fraquil, a defined freshwater medium, was used as a highly reproducible medium to study the behaviour of Lp in water. Adopting a reductionist approach, Fraquil was used to study the impact of temperature, pH and trace metal levels on the survival and subsequent intracellular multiplication of Lp in Acanthamoeba castellanii, a freshwater protozoan and a natural host of Legionella. We show that temperature has a significant impact on the short- and long-term survival of Lp, but that the bacterium retains intracellular multiplication potential for over six months in Fraquil. Moreover, incubation in Fraquil at pH 4.0 resulted in a rapid decline in colony forming units, but was not detrimental to intracellular multiplication. In contrast, variations in trace metal concentrations had no impact on either survival or intracellular multiplication in amoeba. Our data show that Lp is a resilient bacterium in the water environment, remaining infectious to host cells after six months under the nutrient-deprived conditions of Fraquil