Replication of Legionella Pneumophila in Human Cells: Why are We Susceptible?

Legionella pneumophila is the causative agent of Legionnaires’ disease, a serious and often fatal form of pneumonia. The susceptibility to L. pneumophila arises from the ability of this intracellular pathogen to multiply in human alveolar macrophages and monocytes. L. pneumophila also replicates in several professional and non-professional phagocytic human-derived cell lines. With the exception of the A/J mouse strain, most mice strains are restrictive, thus they do not support L. pneumophila replication. Mice lacking the NOD-like receptor Nlrc4 or caspase-1 are also susceptible to L. pneumophila. On the other hand, in the susceptible human hosts, L. pneumophila utilizes several strategies to ensure intracellular replication and protect itself against the host immune system. Most of these strategies converge to prevent the fusion of the L. pneumophila phagosome with the lysosome, inhibiting host cell apoptosis, activating survival pathways, and sequestering essential nutrients for replication and pathogenesis. In this review, we summarize survival mechanisms employed by L. pneumophila to maintain its replication in human cells. In addition, we highlight different human-derived cell lines that support the multiplication of this intracellular bacterium. Therefore, these in vitro models can be applicable and are reproducible when investigating L. pneumophila/phagocyte interactions at the molecular and cellular levels in the human host

Biofilm, a Cozy Structure for <em>Legionella pneumophila</em> Growth and Persistence in the Environment

Legionella pneumophila (L. pneumophila) is the causative agent of Legionnaires’ disease. Transmission to humans is mediated via inhalation of contaminated water droplets. L. pneumophila is widely distributed in man-made water systems, multiple species of protozoa, and nematodes. L. pneumophila persist within multi-species biofilms that cover surfaces within water systems. Virulence, spread, and resistance to biocides are associated with survival of L. pneumophila within multi-organismal biofilm. Outbreaks of Legionellosis are correlated with the existence of L. pneumophila in biofilms, even after the intensive chemical and physical treatments. Several factors negatively or positively modulate the persistence of L. pneumophila within the microbial consortium-containing L. pneumophila. Biofilm-forming L. pneumophila continue to be a public health and economic burden and directly influence the medical and industrial sectors. Diagnosis and hospitalization of patients and prevention protocols cost governments billions of dollars. Dissecting the biological and environmental factors that promote the persistence and physiological adaptation in biofilms can be fundamental to eliminating and preventing the transmission of L. pneumophila. Herein, we review different factors that promote persistence of L. pneumophila within the biofilm consortium, survival strategies used by the bacteria within biofilm community, gene regulation, and finally challenges associated with biofilm resistance to biocides and anti-Legionella treatments

Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation

Gout is characterized by attacks of arthritis with hyperuricemia and monosodium urate (MSU) crystal-induced inflammation within joints. Innate immune responses are the primary drivers for tissue destruction and inflammation in gout. MSU crystals engage the Nlrp3 inflammasome, leading to the activation of caspase-1 and production of IL-1β and IL-18 within gout-affected joints, promoting the influx of neutrophils and monocytes. Here, we show that caspase-11−/− mice and their derived macrophages produce significantly reduced levels of gout-specific cytokines including IL-1β, TNFα, IL-6, and KC, while others like IFNγ and IL-12p70 are not altered. IL-1β induces the expression of caspase-11 in an IL-1 receptor-dependent manner in macrophages contributing to the priming of macrophages during sterile inflammation. The absence of caspase-11 reduced the ability of macrophages and neutrophils to migrate in response to exogenously injected KC in vivo. Notably, in vitro, caspase-11−/− neutrophils displayed random migration in response to a KC gradient when compared to their WT counterparts. This phenotype was associated with altered cofilin phosphorylation. Unlike their wild-type counterparts, caspase-11−/− neutrophils also failed to produce neutrophil extracellular traps (NETs) when treated with MSU. Together, this is the first report demonstrating that caspase-11 promotes neutrophil directional trafficking and function in an acute model of gout. Caspase-11 also governs the production of inflammasome-dependent and -independent cytokines from macrophages. Our results offer new, previously unrecognized functions for caspase-11 in macrophages and neutrophils that may apply to other neutrophil-mediated disease conditions besides gout

Factors Mediating Environmental Biofilm Formation by Legionella pneumophila

Legionella pneumophila (L. pneumophila) is an opportunistic waterborne pathogen and the causative agent for Legionnaires' disease, which is transmitted to humans via inhalation of contaminated water droplets. The bacterium is able to colonize a variety of man-made water systems such as cooling towers, spas, and dental lines and is widely distributed in multiple niches, including several species of protozoa In addition to survival in planktonic phase, L. pneumophila is able to survive and persist within multi-species biofilms that cover surfaces within water systems. Biofilm formation by L. pneumophila is advantageous for the pathogen as it leads to persistence, spread, resistance to treatments and an increase in virulence of this bacterium. Furthermore, Legionellosis outbreaks have been associated with the presence of L. pneumophila in biofilms, even after the extensive chemical and physical treatments. In the microbial consortium-containing L. pneumophila among other organisms, several factors either positively or negatively regulate the presence and persistence of L. pneumophila in this bacterial community. Biofilm-forming L. pneumophila is of a major importance to public health and have impact on the medical and industrial sectors. Indeed, prevention and removal protocols of L. pneumophila as well as diagnosis and hospitalization of patients infected with this bacteria cost governments billions of dollars. Therefore, understanding the biological and environmental factors that contribute to persistence and physiological adaptation in biofilms can be detrimental to eradicate and prevent the transmission of L. pneumophila. In this review, we focus on various factors that contribute to persistence of L. pneumophila within the biofilm consortium, the advantages that the bacteria gain from surviving in biofilms, genes and gene regulation during biofilm formation and finally challenges related to biofilm resistance to biocides and anti-Legionella treatments

The Sphingosine-1-Phosphate Lyase (LegS2) Contributes to the Restriction of Legionella pneumophila in Murine Macrophages.

L. pneumophila is the causative agent of Legionnaires' disease, a human illness characterized by severe pneumonia. In contrast to those derived from humans, macrophages derived from most mouse strains restrict L. pneumophila replication. The restriction of L. pneumophila replication has been shown to require bacterial flagellin, a component of the type IV secretion system as well as the cytosolic NOD-like receptor (NLR) Nlrc4/ Ipaf. These events lead to caspase-1 activation which, in turn, activates caspase-7. Following caspase-7 activation, the phagosome-containing L. pneumophila fuses with the lysosome, resulting in the restriction of L. pneumophila growth. The LegS2 effector is injected by the type IV secretion system and functions as a sphingosine 1-phosphate lyase. It is homologous to the eukaryotic sphingosine lyase (SPL), an enzyme required in the terminal steps of sphingolipid metabolism. Herein, we show that mice Bone Marrow-Derived Macrophages (BMDMs) and human Monocyte-Derived Macrophages (hMDMs) are more permissive to L. pneumophila legS2 mutants than wild-type (WT) strains. This permissiveness to L. pneumophila legS2 is neither attributed to abolished caspase-1, caspase-7 or caspase-3 activation, nor due to the impairment of phagosome-lysosome fusion. Instead, an infection with the legS2 mutant resulted in the reduction of some inflammatory cytokines and their corresponding mRNA; this effect is mediated by the inhibition of the nuclear transcription factor kappa-B (NF-κB). Moreover, BMDMs infected with L. pneumophila legS2 mutant showed elongated mitochondria that resembles mitochondrial fusion. Therefore, the absence of LegS2 effector is associated with reduced NF-κB activation and atypical morphology of mitochondria

Replication of <i>L</i>. <i>pneumophila legS2</i> mutant is not due to defective phagosome-lysosome fusion.

<p>(A) Images of wild-type macrophages not infected (NT) or infected with the type IV secretion mutant <i>dotA</i>, wild-type <i>L</i>. <i>pneumophila</i>, JR32, and the <i>legS2</i> mutant. The first panel presents staining with DAPI, with arrow heads pointing to the bacteria. The second panel shows staining with the lyso tracker. The third panel depicts merged images, with bacteria colocalized with the lysosomal marker. B) The percent of bacteria colocalized with the lyso tracker was scored in 100 infected cells from 3 independent coverslips. The data represents the mean + SD of n = 3.</p

The permissiveness of mice BMDMs to <i>legS2</i> mutant bacteria is independent of caspase-1 or caspase-7 activation.

<p>(A) Caspase-1 KO (casp-1^-/-) macrophages were infected with <i>L</i>. <i>pneumophila</i>, JR32, or <i>legS2</i> with an MOI of 0.5 for 1, 24, 48 and 72 h; then, CFUs were measured at the indicated time points. (B) Levels of IL-1β were detected in supernatants of WT or casp-1^-/- BMDMs were infected with JR32 or the <i>legS2</i> mutant after 24 hr, while WT BMDMs were either not treated (NT) or infected with <i>L</i>. <i>pneumophila</i>, JR32, or <i>legS2</i> mutant bacteria for 2 h. Salmonella infection (Sal) was used as a positive control for caspase-1 or caspase-7 activation. (C) Casp-1 or (D) casp-7 antibodies were used to detect casp-1 and casp-7 activation, respectively, in cell extracts.</p

Infection with the <i>legS2</i> mutant is accompanied by change in mitochondrial morphology.

<p>TEM of BMDMs infected with the JR32 or the <i>legS2</i> mutant. Images were taken from 24 h post-infected cells.</p

The <i>legS2</i> mutant inhibits the NF-κB pathway.

<p>Wild-type macrophages were either not treated (NT) or infected with the JR32, or the <i>legS2</i> mutant for 1, 4, and 8h. Then, nuclear extracts were processed using electrophoretic mobility shift assay (EMSA) to determine NF-κB activation.</p

Replication of <i>legS2</i> mutant is independent of host cell death.

<p>(A) Wild-type BMDMs were not treated (NT) or infected with the type IV secretion mutant <i>dotA</i>, wild-type <i>L</i>. <i>pneumophila</i>, JR32, or <i>legS2</i> mutant for 24h at MOIs of 0.5 and 5. Then, the fold change in LDH release was measured from the overall population of macrophages. The data represents the mean + SD of n = 3. (B) Wild-type C57BL/6 (B6) were not treated (NT) or infected with wild-type <i>L</i>. <i>pneumophila</i>, JR32, <i>legS2</i>, or <i>dotA</i> mutant for 8 h. Salmonella infection (Sal) was used as a positive control for caspase-3 activation. A Western blot with caspase-3 antibody was used to detect casp-3 activation. β-actin was used as a loading control.</p