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. pneumophila-containing vacuoles and autophagosomes share several features, including endoplasmic reticulum (ER)-derived membranes, contributions by the host GTPases Rab1, Arf1 and Sar1, and a final destiny in lysosomes. We discuss morphological, molecular genetic, and immunological data that support the model that, although A/J mouse macrophages efficiently engulf L. pneumophila within autophagosomal membranes, the Type IV effector proteins DrrA/SidM, LidA, and RalF prolong association with the ER. By inhibiting immediately delivery to lysosomes, the bacteria persist in immature autophagosomal vacuoles for a period sufficient to differentiate into an acid-resistant, replicative form. Subsequent secretion of the Type IV effector LepB releases the block to autophagosome maturation, and the adapted progeny continue to replicate within autophagolysosomes. Accordingly, L. pneumophila can be exploited as a genetic tool to analyze the recruitment and function of the macrophage autophagy pathway

SpoT governs Legionella pneumophila differentiation in host macrophages

During its life cycle, Legionella pneumophila alternates between a replicative and a transmissive state. To determine their contributions to L. pneumophila differentiation, the two ppGpp synthetases, RelA and SpoT, were disrupted. Synthesis of ppGpp was required for transmission, as relA spoT mutants were killed during entry to and exit from macrophages. RelA, which senses amino acid starvation induced by serine hydroxamate, is dispensable in macrophages, as relA mutants spread efficiently. SpoT monitors fatty acid biosynthesis (FAB), since following cerulenin treatment, wild-type and relA strains expressed the flaA transmissive gene, but relA spoT mutants did not. As in Escherichia coli , the SpoT response to FAB perturbation likely required an interaction with acyl-carrier protein (ACP), as judged by the failure of the spoT-A413E allele to rescue transmissive trait expression of relA spoT bacteria. Furthermore, SpoT was essential for transmission between macrophages, since secondary infections by relA spoT mutants were restored by induction of spoT , but not relA . To resume replication, ppGpp must be degraded, as mutants lacking spoT hydrolase activity failed to convert from the transmissive to the replicative phase in either bacteriological medium or macrophages. Thus, L. pneumophila requires SpoT to monitor FAB and to alternate between replication and transmission in macrophages.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73309/1/j.1365-2958.2008.06555.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/73309/2/MMI_6555_sm_Figure_S1.pd

Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection.

To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1beta secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection

Novel (p)ppGpp Binding and Metabolizing Proteins of <i>Escherichia coli</i>

The alarmone (p)ppGpp plays pivotal roles in basic bacterial stress responses by increasing tolerance of various nutritional limitations and chemical insults, including antibiotics. Despite intensive studies since (p)ppGpp was discovered over 4 decades ago, (p)ppGpp binding proteins have not been systematically identified in Escherichia coli. We applied DRaCALA (differential radial capillary action of ligand assay) to identify (p)ppGpp-protein interactions. We discovered 12 new (p)ppGpp targets in E. coli that, based on their physiological functions, could be classified into four major groups, involved in (i) purine nucleotide homeostasis (YgdH), (ii) ribosome biogenesis and translation (RsgA, Era, HflX, and LepA), (iii) maturation of dehydrogenases (HypB), and (iv) metabolism of (p)ppGpp (MutT, NudG, TrmE, NadR, PhoA, and UshA). We present a comprehensive and comparative biochemical and physiological characterization of these novel (p)ppGpp targets together with a comparative analysis of relevant, known (p)ppGpp binding proteins. Via this, primary targets of (p)ppGpp in E. coli are identified. The GTP salvage biosynthesis pathway and ribosome biogenesis and translation are confirmed as targets of (p)ppGpp that are highly conserved between E. coli and Firmicutes. In addition, an alternative (p)ppGpp degradative pathway, involving NudG and MutT, was uncovered. This report thus significantly expands the known cohort of (p)ppGpp targets in E. coli

Facilitation of Psychiatric Advance Directives by Peers and Clinicians on Assertive Community Treatment Teams

OBJECTIVE: Psychiatric advance directives (PADs) provide a legal mechanism for competent adults to document care preferences and authorize a surrogate to make treatment decisions. In a controlled research setting, an evidence-based intervention, the facilitated psychiatric advance directive (FPAD), was previously shown to overcome most barriers to PAD completion. This study examined implementation of the FPAD intervention in usual care settings as delivered by peer support specialists and nonpeer clinicians on assertive community treatment (ACT) teams. METHODS: A total of 145 ACT consumers were randomly assigned, within teams, to FPAD with facilitation by either a peer (N=71) or a clinician (N=74). Completion rates and PAD quality were compared with the previous study's standard and across facilitator type. Logistic regression was used to estimate effects on the likelihood of PAD completion. RESULTS: The completion rate of 50% in the intent-to-treat sample (N=145) was somewhat inferior to the prior standard (61%), but the rate of 58% for the retained sample (those who completed a follow-up interview, N=116) was not significantly different from the standard. Rates for peers and clinicians did not differ significantly from each other for either sample. PAD quality was similar to that achieved in the prior study. Four consumer variables predicted completion: independent living status, problematic substance use, length of time served by the ACT team, and no perceived unmet need for hospitalization in crisis. CONCLUSIONS: Peers and clinicians can play a crucial role in increasing the number of consumers with PADs, an important step toward improving implementation of PADs in mental health care

Criminal Justice and Suicide Outcomes with Indiana's Risk-Based Gun Seizure Law

This article examines the application and effectiveness of a 2006 Indiana law designed to prevent gun violence by authorizing police officers to separate firearms from persons who present imminent or future risk of injury to self or others, or display a propensity for violent or emotionally unstable conduct. A court hearing is held to determine ongoing risk in these cases; a judge decides whether to return the seized firearms or retain them for up to five years. The study examines the frequency of criminal arrest as well as suicide outcomes for 395 gun-removal actions in Indiana. Fourteen individuals (3.5%) died from suicide, seven (1.8%) using a firearm. The study population's annualized suicide rate was about 31 times higher than that of the general adult population in Indiana, demonstrating that the law is being applied to a population genuinely at high risk. By extrapolating information on the case fatality rate for different methods of suicide, we calculated that one life was saved for every 10 gun-removal actions, similar to results of a previous study in Connecticut. Perspectives from key stakeholders are also presented along with implications for gun policy reform and implementation

Enhancing career development of postdoctoral trainees: act locally and beyond

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149264/1/tjp13462.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149264/2/tjp13462_am.pd

Prevalence of Infection-Competent Serogroup 6 \u3cem\u3eLegionella pneumophila\u3c/em\u3e 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 Legionella pneumophila 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. Common to one environmental and four clinical samples were strains of L. pneumophila SG1 and ST1, the most prevalent ST worldwide. Among the Flint premise plumbing isolates, 14 of 16 strains were of ST367 and ST461, two closely related SG6 strain types isolated previously from patients and corresponding environmental samples. Each of the representative SG1 clinical strains and SG6 environmental isolates from Southeast Michigan infected and survived within macrophage cultures at least as well as a virulent laboratory strain, as judged by microscopy and by enumerating CFU. Likewise, 72 h after infection, the yield of viable-cell counts increased \u3e 100-fold for each of the representative SG1 clinical isolates, Flint premise plumbing SG6 ST367 and -461 isolates, and two Detroit residential isolates. We verified by immunostaining that SG1-specific antibody does not cross-react with the SG6 L. pneumophila environmental strains. Because the widely used urinary antigen diagnostic test does not readily detect non-SG1 L. pneumophila, Legionnaires’ disease caused by SG6 L. pneumophila is likely underreported worldwide