Cryo-EM reveals new species-specific proteins and symmetry elements in the Dot/Icm T4SS.

is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.

Functional characterization of the HMP-P synthase of Legionella pneumophila (Lpg1565).

The production of the pyrimidine moiety in thiamine synthesis, 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P), has been described to proceed through the Thi5-dependent pathway in Saccharomyces cerevisiae and other yeast. Previous work found that ScThi5 functioned poorly in a heterologous context. Here we report a bacterial ortholog to the yeast HMP-P synthase (Thi5) was necessary for HMP synthesis in Legionella pneumophila.

Structural analysis of the Dot/Icm type IV secretion system core complex.

is an opportunistic pathogen that causes the potentially fatal pneumonia Legionnaires' Disease. This infection and subsequent pathology require the Dot/Icm Type IV Secretion System (T4SS) to deliver effector proteins into host cells. Compared to prototypical T4SSs, the Dot/Icm assembly is much larger, containing ~27 different components including a core complex reported to be composed of five proteins: DotC, DotD, DotF, DotG, and DotH.

How defend their turf.

Communities of bacteria that cause Legionnaires' disease repel other bacteria by secreting an acid called HGA.

A Two-Component System That Modulates Cyclic di-GMP Metabolism Promotes Legionella pneumophila Differentiation and Viability in Low-Nutrient Conditions.

During its life cycle, the environmental pathogen alternates between a replicative and transmissive cell type when cultured in broth, macrophages, or amoebae. Within a protozoan host, further differentiates into the hardy cell type known as the mature infectious form (MIF). The second messenger cyclic di-GMP coordinates lifestyle changes in many bacterial species, but its role in the life cycle is less understood.

Symbiont-Mediated Defense against Legionella pneumophila in Amoebae.

is an important opportunistic pathogen for which environmental reservoirs are crucial for the infection of humans. In the environment, free-living amoebae represent key hosts providing nutrients and shelter for highly efficient intracellular proliferation of , which eventually leads to lysis of the protist. However, the significance of other bacterial players for ecology is poorly understood.

Prevalence of Infection-Competent Serogroup 6 within Premise Plumbing in Southeast Michigan.

Coinciding with major changes to its municipal water system, Flint, MI, endured Legionnaires' disease outbreaks in 2014 and 2015. By sampling premise plumbing in Flint in the fall of 2016, we found that 12% of homes harbored legionellae, a frequency similar to that in residences in neighboring areas. To evaluate the genetic diversity of in Southeast Michigan, we determined the sequence type (ST) and serogroup (SG) of the 18 residential isolates from Flint and Detroit, MI, and the 33 clinical isolates submitted by hospitals in three area counties in 2013 to 2016.

Assessment of the Legionnaires' disease outbreak in Flint, Michigan.

The 2014-2015 Legionnaires' disease (LD) outbreak in Genesee County, MI, and the outbreak resolution in 2016 coincided with changes in the source of drinking water to Flint's municipal water system. Following the switch in water supply from Detroit to Flint River water, the odds of a Flint resident presenting with LD increased 6.3-fold (95% CI: 2.

Renitence vacuoles facilitate protection against phagolysosomal damage in activated macrophages.

As professional phagocytes, macrophages are susceptible to endolysosomal membrane damage inflicted by the pathogens and noxious particles they ingest. Whether macrophages have mechanisms for limiting such damage is not well understood. Previously, we reported a phenomenon, termed "inducible renitence," in which lipopolysaccharide (LPS) activation of macrophages protected their endolysosomes against damage initiated by the phagocytosis of silica beads.

csrT Represents a New Class of csrA-Like Regulatory Genes Associated with Integrative Conjugative Elements of Legionella pneumophila.

Unlabelled: Bacterial evolution is accelerated by mobile genetic elements. To spread horizontally and to benefit the recipient bacteria, genes encoded on these elements must be properly regulated. Among the legionellae are multiple integrative conjugative elements (ICEs) that each encode a paralog of the broadly conserved regulator csrA.

csrR, a Paralog and Direct Target of CsrA, Promotes Legionella pneumophila Resilience in Water.

Unlabelled: Critical to microbial versatility is the capacity to express the cohort of genes that increase fitness in different environments. Legionella pneumophila occupies extensive ecological space that includes diverse protists, pond water, engineered water systems, and mammalian lung macrophages. One mechanism that equips this opportunistic pathogen to adapt to fluctuating conditions is a switch between replicative and transmissive cell types that is controlled by the broadly conserved regulatory protein CsrA.

Integrative conjugative element ICE-βox confers oxidative stress resistance to Legionella pneumophila in vitro and in macrophages.

ABSTRACT Integrative conjugative elements (ICEs) are mobile blocks of DNA that can contribute to bacterial evolution by self-directed transmission of advantageous traits. Here, we analyze the activity of a putative 65-kb ICE harbored by Legionella pneumophila using molecular genetics, conjugation assays, a phenotype microarray screen, and macrophage infections. The element transferred to a naive L.

Nutrient salvaging and metabolism by the intracellular pathogen Legionella pneumophila.

The Gram-negative bacterium Legionella pneumophila is ubiquitous in freshwater environments as a free-swimming organism, resident of biofilms, or parasite of protozoa. If the bacterium is aerosolized and inhaled by a susceptible human host, it can infect alveolar macrophages and cause a severe pneumonia known as Legionnaires' disease. A sophisticated cell differentiation program equips L.

The phtC-phtD locus equips Legionella pneumophila for thymidine salvage and replication in macrophages.

The phagosomal transporter (Pht) family of the major facilitator superfamily (MFS) is encoded by phylogenetically related intracellular gammaproteobacteria, including the opportunistic pathogen Legionella pneumophila. The location of the pht genes between the putative thymidine kinase (tdk) and phosphopentomutase (deoB) genes suggested that the phtC and phtD loci contribute to thymidine salvage in L. pneumophila.

Inflammasome components coordinate autophagy and pyroptosis as macrophage responses to infection.

Unlabelled: When microbes contaminate the macrophage cytoplasm, leukocytes undergo a proinflammatory death that is initiated by nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) that bind and activate caspase-1. We report that these inflammasome components also regulate autophagy, a vesicular pathway to eliminate cytosolic debris. In response to infection with flagellate Legionella pneumophila, C57BL/6J mouse macrophages equipped with caspase-1 and the NLR proteins NAIP5 and NLRC4 stimulated autophagosome turnover.

Nicotinic acid modulates Legionella pneumophila gene expression and induces virulence traits.

In response to environmental fluctuations or stresses, bacteria can activate transcriptional and phenotypic programs to coordinate an adaptive response. The intracellular pathogen Legionella pneumophila converts from a noninfectious replicative form to an infectious transmissive form when the bacterium encounters alterations in either amino acid concentrations or fatty acid biosynthesis. Here, we report that L.

Constructing unmarked gene deletions in Legionella pneumophila.

The ability to construct recombinant alleles efficiently in strains of interest, particularly unmarked deletions that reduce the potential for polar effects, is essential to studies of both pathogenesis and basic bacterial physiology. Here we describe a three-phase approach for generating unmarked deletions in Legionella pneumophila by constructing a mutant allele in E. coli using λ-Red recombination, so-called recombineering; transferring the allele onto the L.

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding.

ppGpp: magic beyond RNA polymerase.

During stress, bacteria undergo extensive physiological transformations, many of which are coordinated by ppGpp. Although ppGpp is best known for enhancing cellular resilience by redirecting the RNA polymerase (RNAP) to certain genes, it also acts as a signal in many other cellular processes in bacteria. After a brief overview of ppGpp biosynthesis and its impact on promoter selection by RNAP, we discuss how bacteria exploit ppGpp to modulate the synthesis, stability or activity of proteins or regulatory RNAs that are crucial in challenging environments, using mechanisms beyond the direct regulation of RNAP activity.

Autophagy-mediated dendritic cell activation is essential for innate cytokine production and APC function with respiratory syncytial virus responses.

The regulation of innate immune responses during viral infection is a crucial step to promote antiviral reactions. Recent studies have drawn attention to a strong relationship of pathogen-associated molecular pattern recognition with autophagy for activation of APC function. Our initial observations indicated that autophagosomes formed in response to respiratory syncytial virus (RSV) infection of dendritic cells (DC).

Secrets of a successful pathogen: legionella resistance to progression along the autophagic pathway.

To proliferate within phagocytes, Legionella pneumophila relies on Type IV secretion to modulate host cellular pathways. Autophagy is an evolutionarily conserved degradative pathway that captures and transfers a variety of microbes to lysosomes. Biogenesis of L.