Intracellular Bacterial Pathogen Research Articles

TNF licenses macrophages to undergo rapid caspase-1, -11, and -8-mediated cell death that restricts Legionella pneumophila infection.

The inflammatory cytokine tumor necrosis factor (TNF) is necessary for host defense against many intracellular pathogens, including Legionella pneumophila. Legionella causes the severe pneumonia Legionnaires' disease and predominantly affects individuals with a suppressed immune system, including those receiving therapeutic TNF blockade to treat autoinflammatory disorders. TNF induces pro-inflammatory gene expression, cellular proliferation, and survival signals in certain contexts, but can also trigger programmed cell death in others. It remains unclear, however, which of the pleiotropic functions of TNF mediate control of intracellular bacterial pathogens like Legionella. In this study, we demonstrate that TNF signaling licenses macrophages to die rapidly in response to Legionella infection. We find that TNF-licensed cells undergo rapid gasdermin-dependent, pyroptotic death downstream of inflammasome activation. We also find that TNF signaling upregulates components of the inflammasome response, and that the caspase-11-mediated non-canonical inflammasome is the first inflammasome to be activated, with caspase-1 and caspase-8 mediating delayed pyroptotic death. We find that all three caspases are collectively required for optimal TNF-mediated restriction of bacterial replication in macrophages. Furthermore, caspase-8 is required for control of pulmonary Legionella infection. These findings reveal a TNF-dependent mechanism in macrophages for activating rapid cell death that is collectively mediated by caspases-1, -8, and -11 and subsequent restriction of Legionella infection.

Peptidoglycan deacetylation controls type IV secretion and the intracellular survival of the bacterial pathogen Legionella pneumophila

Peptidoglycan is a critical component of the bacteria cell envelope. Remodeling of the peptidoglycan is required for numerous essential cellular processes and has been linked to bacterial pathogenesis. Peptidoglycan deacetylases that remove the acetyl group of the N-acetylglucosamine (NAG) subunit protect bacterial pathogens from immune recognition and digestive enzymes secreted at the site of infection. However, the full extent of this modification on bacterial physiology and pathogenesis is not known. Here, we identify a polysaccharide deacetylase of the intracellular bacterial pathogen Legionella pneumophila and define a two-tiered role for this enzyme in Legionella pathogenesis. First, NAG deacetylation is important for the proper localization and function of the Type IVb secretion system, linking peptidoglycan editing to the modulation of host cellular processes through the action of secreted virulence factors. As a consequence, the Legionella vacuole mis-traffics along the endocytic pathway to the lysosome, preventing the formation of a replication permissive compartment. Second, within the lysosome, the inability to deacetylate the peptidoglycan renders the bacteria more sensitive to lysozyme-mediated degradation, resulting in increased bacterial death. Thus, the ability to deacetylate NAG is important for bacteria to persist within host cells and in turn, Legionella virulence. Collectively, these results expand the function of peptidoglycan deacetylases in bacteria, linking peptidoglycan editing, Type IV secretion, and the intracellular fate of a bacterial pathogen.

A Homing Missile-Like Nanotherapeutic with Single-Atom Catalytic Sites for In Situ Elimination of Intracellular Bacterial Pathogens.

Intracellular bacterial pathogens hiding in host cells tolerate the innate immune system and high-dose antibiotics, resulting in recurrent infections that are difficult to treat. Herein, a homing missile-like nanotherapeutic (FeSAs@Sa.M) composed of a single-atom iron nanozyme (FeSAs) core coated with infected macrophage membrane (Sa.M) is developed for in situ elimination of intracellular methicillin-resistant S. aureus (MRSA). Mechanically, the FeSAs@Sa.M initially binds to the extracellular MRSA via the bacterial recognition ability of the Sa.M component. Subsequently, the FeSAs@Sa.M can be transported to the intracellular MRSA-located regions in the host cell like a homing missile under the guidance of the extracellular MRSA to which it is attached, generating highly toxic reactive oxygen species (ROS) for intracellular MRSA killing via the enzymatic activities of the FeSAs core. The FeSAs@Sa.M is far superior to FeSAs in killing intracellular MRSA, proposing a feasible strategy for treating intracellular infections by in situ generating ROS in bacterial residing regions.

Genome organization and genomics in Chlamydia: whole genome sequencing increases understanding ofchlamydial virulence, evolution, and phylogeny.

The genus Chlamydia contains important obligate intracellular bacterial pathogens to humans and animals, including C. trachomatis and C. pneumoniae. Since 1998, when the first Chlamydia genome was published, our understanding of how these microbes interact, evolved and adapted to different intracellular host environments has been transformed due to the expansion of chlamydial genomes. This review explores the current state of knowledge in Chlamydia genomics and how whole genome sequencing has revolutionised our understanding of Chlamydia virulence, evolution, and phylogeny over the past two and a half decades. This review will also highlight developments in multi-omics and other approaches that have complemented whole genome sequencing to advance knowledge of Chlamydia pathogenesis and future directions for chlamydial genomics.

Chronically hypertensive transgenic mice expressing human AT1R haplotype-I exhibit increased susceptibility to Francisella tularensis.

Age-related illnesses, including hypertension and accompanying metabolic disorders, compromise immunity and exacerbate infection-associated fatalities. Renin-angiotensin system (RAS) is the key mechanism that controls blood pressure. Upregulation of RAS through angiotensin receptor type 1 (AT1R), a G-protein coupled receptor, contributes to the pathophysiological consequences leading to vascular remodeling, hypertension, and end-organ damage. Genetic variations that increase the expression of human AT1R may cause the above pathological outcomes associated with hypertension. Previously we have shown that our chronically hypertensive transgenic (TG) mice containing the haplotype-I variant (Hap-I, hypertensive genotype) of human AT1R (hAT1R) gene are more prone to develop the metabolic syndrome-related disorders as compared to the TG mice containing the haplotype-II variant (Hap-II, normotensive genotype). Since aging and an increased risk of hypertension can impact multiple organ systems in a complex manner, including susceptibility to various infections, the current study investigated the susceptibility and potential effect of acute bacterial infection using a Gram-negative intracellular bacterial pathogen, Francisella tularensis in our hAT1R TG mice. Our results show that compared to Hap-II, F. tularensis-infected aged Hap-I TG mice have significantly higher mortality post-infection, higher bacterial load and lung pathology, elevated inflammatory cytokines and altered gene expression profile favoring hypertension and inflammation. Consistent with worsened phenotype in aged Hap-I mice post-Francisella infection, gene expression profiles from their lungs revealed significantly altered expression of more than 1,400 genes. Furthermore, bioinformatics analysis identified genes associated with RAS and IFN-γ pathways regulating blood pressure and inflammation. These studies demonstrate that haplotype-dependent over-expression of the hAT1R gene leads to enhanced susceptibility and lethality due to F. tularensis LVS infection, which gets aggravated in aged animals. Clinically, these findings will help in exploring the role of AT1R-induced hypertension and enhanced susceptibility to infection-related respiratory diseases.

The Unique N-Terminal Domain of Chlamydial Bactofilin Mediates Its Membrane Localization and Ring-Forming Properties.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen. In evolving to the intracellular niche, Chlamydia has reduced its genome size compared to other bacteria and, as a consequence, has a number of unique features. For example, Chlamydia engages the actin-like protein MreB, rather than the tubulin-like protein FtsZ, to direct peptidoglycan (PG) synthesis exclusively at the septum of cells undergoing polarized cell division. Interestingly, Chlamydia possesses another cytoskeletal element-a bactofilin ortholog, BacA. Recently, we reported BacA is a cell size-determining protein that forms dynamic membrane-associated ring structures in Chlamydia that have not been observed in other bacteria with bactofilins. Chlamydial BacA possesses a unique N-terminal domain, and we hypothesized this domain imparts the membrane-binding and ring-forming properties of BacA. We show that different truncations of the N terminus result in distinct phenotypes: removal of the first 50 amino acids (ΔN50) results in large ring structures at the membrane whereas removal of the first 81 amino acids (ΔN81) results in an inability to form filaments and rings and a loss of membrane association. Overexpression of the ΔN50 isoform altered cell size, similar to loss of BacA, suggesting that the dynamic properties of BacA are essential for the regulation of cell size. We further show that the region from amino acid 51 to 81 imparts membrane association as appending it to green fluorescent protein (GFP) resulted in the relocalization of GFP from the cytosol to the membrane. Overall, our findings suggest two important functions for the unique N-terminal domain of BacA and help explain its role as a cell size determinant. IMPORTANCE Bacteria use a variety of filament-forming cytoskeletal proteins to regulate and control various aspects of their physiology. For example, the tubulin-like FtsZ recruits division proteins to the septum whereas the actin-like MreB recruits peptidoglycan (PG) synthases to generate the cell wall in rod-shaped bacteria. Recently, a third class of cytoskeletal protein has been identified in bacteria-bactofilins. These proteins have been primarily linked to spatially localized PG synthesis. Interestingly, Chlamydia, an obligate intracellular bacterium, does not have PG in its cell wall and yet possesses a bactofilin ortholog. In this study, we characterize a unique N-terminal domain of chlamydial bactofilin and show that this domain controls two important functions that affect cell size: its ring-forming and membrane-associating properties.

A single chlamydial protein reshapes the plasma membrane and serves as recruiting platform for central endocytic effector proteins

Uptake of obligate intracellular bacterial pathogens into mammalian epithelial cells is critically dependent on modulation of the host’s endocytic machinery. It is an open question how the invading pathogens generate a membrane-bound vesicle appropriate to their size. This requires extensive deformation of the host plasma membrane itself by pathogen-derived membrane-binding proteins, accompanied by substantial F-actin-based forces to further expand and finally pinch off the vesicle. Here we show that upon adhesion to the host cell, the human pathogenic bacterium Chlamydia pneumoniae secretes the scaffolding effector protein CPn0677, which binds to the inner leaflet of the invaginating host’s PM, induces inwardly directed, negative membrane curvature, and forms a recruiting platform for the membrane-deforming BAR-domain containing proteins Pacsin and SNX9. In addition, while bound to the membrane, CPn0677 recruits monomeric G-actin, and its C-terminal region binds and activates N-WASP, which initiates branching actin polymerization via the Arp2/3 complex. Together, these membrane-bound processes enable the developing endocytic vesicle to engulf the infectious elementary body, while the associated actin network generates the forces required to reshape and detach the nascent vesicle from the PM. Thus, Cpn0677 (now renamed SemD) acts as recruiting platform for central components of the endocytic machinery during uptake of chlamydia.

Elucidating the role of the host protein, FBXO22, in the infection of macrophages with the zoonotic bacterial pathogen, Brucella.

Abstract Brucella spp. are facultative intracellular bacterial pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes. Successful invasion and chronic intracellular persistence indicate that Brucella manipulates various host cellular processes to create a replication permissive niche. The essential mechanisms and host factors that support the invasion and intracellular replication of Brucella remain poorly understood. RNA interference (RNAi) is a potent molecular tool for understanding gene function by downregulating the target gene expression through mRNA degradation. We used siRNA-based screening to identify the host factors that are involved in Brucella-macrophage interaction. Macrophages were treated with gene specific siRNAs, followed by infection with B. neotomae/B. melitensis and quantification of intracellular Brucella. The screening identified several host factors that are involved in the Brucella infection of macrophages. Subsequently, we performed detailed characterization of one of the identified host proteins, FBXO22. Downregulation of FBXO22 resulted in diminished uptake of Brucella into macrophages, which was dependent on NF-κB-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella-infected macrophages that resulted in induction of phagocytic receptors and enhanced production of pro-inflammatory cytokines through NF-κB. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella, including the anti-inflammatory proteins, for degradation through the SCF complex. Funding from Department of Biotechnology (DBT) (BT/PR12301/ADV/90/176/2014)

Bacterial blockade of host protein synthesis triggers cell death in dendritic cells to restrict Legionella pneumophilainfection

Abstract Legionella pneumophila is a facultative intracellular bacterial pathogen that is responsible for a pneumonia called Legionnaire’s disease. Legionella invades cells by using a type IV secretion system that delivers effector proteins into the host cell cytosol to facilitate its intracellular survival and replication. Macrophages serve as the primary replicative niche for Legionella. In contrast, dendritic cells (DCs) have been shown to rapidly restrict Legionella infection by activating cell death. The host and bacterial factors that induce this response remain elusive. We found that DCs undergo caspase-dependent cell death and that this response is triggered by a subset of secreted effectors that block host protein synthesis. Specifically, we show that these effectors decrease the levels of pro-survival proteins in DCs, thus leaving cells poised to undergo cell death. Altogether, our data show that pro-survival molecules in DCs serve as guard proteins that detect pathogenic activities to limit bacterial replication. Supported by a National Science Foundation Graduate Research Fellowship Program (DGE-2236662)

The emerging roles of long non-coding RNA in host immune response and intracellular bacterial infections.

The long non-coding RNAs (lncRNAs) are evolutionarily conserved classes of non-coding regulatory transcripts of > 200 nucleotides in length. They modulate several transcriptional and post-transcriptional events in the organism. Depending on their cellular localization and interactions, they regulate chromatin function and assembly; and alter the stability and translation of cytoplasmic mRNAs. Although their proposed range of functionality remains controversial, there is increasing research evidence that lncRNAs play a regulatory role in the activation, differentiation and development of immune signaling cascades; microbiome development; and in diseases such as neuronal and cardiovascular disorders; cancer; and pathogenic infections. This review discusses the functional roles of different lncRNAs in regulation of host immune responses, signaling pathways during host-microbe interaction and infection caused by obligate intracellular bacterial pathogens. The study of lncRNAs is assuming significance as it could be exploited for development of alternative therapeutic strategies for the treatment of severe and chronic pathogenic infections caused by Mycobacterium, Chlamydia and Rickettsia infections, as well as commensal colonization. Finally, this review summarizes the translational potential of lncRNA research in development of diagnostic and prognostic tools for human diseases.

Host A-to-I RNA editing signatures in intracellular bacterial and single-strand RNA viral infections.

Microbial infection is accompanied by remodeling of the host transcriptome. Involvement of A-to-I RNA editing has been reported during viral infection but remains to be elucidated during intracellular bacterial infections. Herein we analyzed A-to-I RNA editing during intracellular bacterial infections based on 18 RNA-Seq datasets of 210 mouse samples involving 7 tissue types and 8 intracellular bacterial pathogens (IBPs), and identified a consensus signature of RNA editing for IBP infections, mainly involving neutrophil-mediated innate immunity and lipid metabolism. Further comparison of host RNA editing patterns revealed remarkable similarities between pneumonia caused by IBPs and single-strand RNA (ssRNA) viruses, such as altered editing enzyme expression, editing site numbers, and levels. In addition, functional enrichment analysis of genes with RNA editing highlighted that the Rab GTPase family played a common and vital role in the host immune response to IBP and ssRNA viral infections, which was indicated by the consistent up-regulated RNA editing of Ras-related protein Rab27a. Nevertheless, dramatic differences between IBP and viral infections were also observed, and clearly distinguished the two types of intracellular infections. Our study showed transcriptome-wide host A-to-I RNA editing alteration during IBP and ssRNA viral infections. By identifying and comparing consensus signatures of host A-to-I RNA editing, our analysis implicates the importance of host A-to-I RNA editing during these infections and provides new insights into the diagnosis and treatment of infectious diseases.

Shigella IpaH9.8 limits GBP1-dependent LPS release from intracytosolic bacteria to suppress caspase-4 activation.

Pyroptosis is an inflammatory form of cell death induced upon recognition of invading microbes. During an infection, pyroptosis is enhanced in interferon-gamma-exposed cells via the actions of members of the guanylate-binding protein (GBP) family. GBPs promote caspase-4 (CASP4) activation by enhancing its interactions with lipopolysaccharide (LPS), a component of the outer envelope of Gram-negative bacteria. Once activated, CASP4 promotes the formation of noncanonical inflammasomes, signaling platforms that mediate pyroptosis. To establish an infection, intracellular bacterial pathogens, like Shigella species, inhibit pyroptosis. The pathogenesis of Shigella is dependent on its type III secretion system, which injects ~30 effector proteins into host cells. Upon entry into host cells, Shigella are encapsulated by GBP1, followed by GBP2, GBP3, GBP4, and in some cases, CASP4. It has been proposed that the recruitment of CASP4 to bacteria leads to its activation. Here, we demonstrate that two Shigella effectors, OspC3 and IpaH9.8, cooperate to inhibit CASP4-mediated pyroptosis. We show that in the absence of OspC3, an inhibitor of CASP4, IpaH9.8 inhibits pyroptosis via its known degradation of GBPs. We find that, while some LPS is present within the host cell cytosol of epithelial cells infected with wild-type Shigella, in the absence of IpaH9.8, increased amounts are shed in a GBP1-dependent manner. Furthermore, we find that additional IpaH9.8 targets, likely GBPs, promote CASP4 activation, even in the absence of GBP1. These observations suggest that by boosting LPS release, GBP1 provides CASP4-enhanced access to cytosolic LPS, thus promoting host cell death via pyroptosis.

Computational Modeling of the Chlamydial Developmental Cycle Reveals a Potential Role for Asymmetric Division.

Chlamydia trachomatis is an obligate intracellular bacterium that progresses through an essential multicell form developmental cycle. Infection of the host is initiated by the elementary body (EB). Once in the host, the EB cell differentiates into the noninfectious, but replication-competent, reticulate body, or RB. After multiple rounds of replication, RBs undergo secondary differentiation, eventually producing newly infectious EBs. Here, we generated paired cell-type promoter reporter constructs and determined the kinetics of the activities of the euo, hctA, and hctB promoters. The paired constructs revealed that the developmental cycle produces at least three phenotypically distinct cell types, the RB (euoprom+), intermediate body (IB; hctAprom+), and EB (hctBprom+). The kinetic data from the three dual-promoter constructs were used to generate two computational agent-based models to reproduce the chlamydial developmental cycle. Both models simulated EB germination, RB amplification, IB formation, and EB production but differed in the mechanism that generated the IB. The direct conversion and the asymmetric production models predicted different behaviors for the RB population, which were experimentally testable. In agreement with the asymmetric production model, RBs acted as stem cells after the initial amplification stage, producing one IB and self-renewing after every division. We also demonstrated that IBs are a transient cell population, maturing directly into EBs after formation without the need for cell division. The culmination of these results suggests that the developmental cycle can be described by a four-stage model, EB germination, RB amplification/maturation, IB production, and EB formation. IMPORTANCE Chlamydia trachomatis is an obligate intracellular bacterial pathogen responsible for both ocular and sexually transmitted infections. All Chlamydiae are reliant on a complex developmental cycle, consisting of both infectious and noninfectious cell forms. The EB cell form initiates infection, whereas the RB cell replicates. The infectious cycle requires both cell types, as RB replication increases the cell population while EB formation disseminates the infection to new hosts. The mechanisms of RB-to-EB development are largely unknown. Here, we developed unique dual promoter reporters and used live-cell imaging and confocal microscopy to visualize the cycle at the single-cell and kinetic levels. These data were used to develop and test two agent-based models, simulating either direct conversion of RBs to EBs or production of EBs via asymmetric RB division. Our results suggest that RBs mature into a stem cell-like population producing intermediate cell forms through asymmetric division, followed by maturation of the intermediate cell type into the infectious EB. Ultimately, a more complete mechanistic understanding of the developmental cycle will lead to novel therapeutics targeting cell type development to eliminate chlamydial dissemination.

Ehrlichia effector SLiM-icry: Artifice of cellular subversion.

As an obligately intracellular bacterial pathogen that selectively infects the mononuclear phagocyte, Ehrlichia chaffeensis has evolved sophisticated mechanisms to subvert innate immune defenses. While the bacterium accomplishes this through a variety of mechanisms, a rapidly expanding body of evidence has revealed that E. chaffeensis has evolved survival strategies that are directed by the versatile, intrinsically disordered, 120 kDa tandem repeat protein (TRP120) effector. E. chaffeensis establishes infection by manipulating multiple evolutionarily conserved cellular signaling pathways through effector-host interactions to subvert innate immune defenses. TRP120 activates these pathways using multiple functionally distinct, repetitive, eukaryote-mimicking short linear motifs (SLiMs) located within the tandem repeat domain that have evolved in nihilo. Functionally, the best characterized TRP120 SLiMs mimic eukaryotic ligands (SLiM-icry) to engage pathway-specific host receptors and activate cellular signaling, thereby repurposing these pathways to promote infection. Moreover, E. chaffeensis TRP120 contains SLiMs that are targets of post-translational modifications such as SUMOylation in addition to many other validated SLiMs that are curated in the eukaryotic linear motif (ELM) database. This review will explore the extracellular and intracellular roles TRP120 SLiM-icry plays during infection - mediated through a variety of SLiMs - that enable E. chaffeensis to subvert mononuclear phagocyte innate defenses.

The Chlamydia trachomatis IncM Protein Interferes with Host Cell Cytokinesis, Centrosome Positioning, and Golgi Distribution and Contributes to the Stability of the Pathogen-Containing Vacuole.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes ocular and urogenital infections in humans. The ability of C. trachomatis to grow intracellularly in a pathogen-containing vacuole (known as an inclusion) depends on chlamydial effector proteins transported into the host cell by a type III secretion system. Among these effectors, several inclusion membrane proteins (Incs) insert in the vacuolar membrane. Here, we show that human cell lines infected by a C. trachomatis strain deficient for Inc CT288/CTL0540 (renamed IncM) displayed less multinucleation than when infected by IncM-producing strains (wild type or complemented). This indicated that IncM is involved in the ability of Chlamydia to inhibit host cell cytokinesis. The capacity of IncM to induce multinucleation in infected cells was shown to be conserved among its chlamydial homologues and appeared to require its two larger regions predicted to be exposed to the host cell cytosol. C. trachomatis-infected cells also displayed IncM-dependent defects in centrosome positioning, Golgi distribution around the inclusion, and morphology and stability of the inclusion. The altered morphology of inclusions containing IncM-deficient C. trachomatis was further affected by depolymerization of host cell microtubules. This was not observed after depolymerization of microfilaments, and inclusions containing wild-type C. trachomatis did not alter their morphology upon depolymerization of microtubules. Overall, these findings suggest that IncM may exert its effector function by acting directly or indirectly on host cell microtubules.

Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderiapseudomallei.

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se.

Legionella longbeachae Regulates the Association of Polyubiquitinated Proteins on Bacterial Phagosome with Multiple Deubiquitinases.

Legionella spp. are the causative agents of a severe pneumonia known as Legionnaires' disease. Upon being engulfed by host cells, these environmental bacteria replicate intracellularly in a plasma membrane-derived niche termed Legionella-containing vacuole (LCV) in a way that requires the defective in organelle trafficking/intracellular multiplication (Dot/Icm) protein transporter. Our understanding of interactions between Legionella and its hosts was mostly based on studies of Legionella pneumophila. In this study, we found that the LCVs created by virulent Legionella longbeachae are similarly decorated by polyubiquitinated proteins to those formed by L. pneumophila and that the ubiquitin-proteasome system (UPS) is required for optimal intracellular growth of L. longbeachae. Furthermore, we utilized bioinformatics methods and the ubiquitin-vinylmethyl ester probe to obtain potential deubiquitinases (DUBs) encoded by L. longbeachae. These efforts led to the identification of 9 L. longbeachae DUBs that displayed distinct specificity toward ubiquitin chain types. Among these, LLO_1014 and LLO_2238 are associated with the LCVs and impact the accumulation of polyubiquitinated species on the bacterial phagosome. Moreover, LLO_1014 and LLO_2238 could fully restore the phenotypes associated with Δceg23 (lotB) and Δlem27 (lotC) mutants of L. pneumophila, indicating that these DUBs have similar functions. Together, these results reveal that L. longbeachae uses multiple DUBs to construct an intracellular niche for its replication. IMPORTANCE Legionella spp. are opportunistic intracellular bacterial pathogens that cause Legionnaires' disease. Legionella utilizes the Dot/Icm type IV secretion system to deliver effector protein into host cells to modulate various cellular functions. At least 26 L. pneumophila effectors are known to hijack the host ubiquitin system via diverse mechanisms. L. longbeachae is the second leading cause of Legionnaires' disease worldwide. However, our knowledge about the interactions between L. longbeachae and its hosts is very limited. Here, we found that, similar to L. pneumophila infection, the host ubiquitin proteasome system is also important for the intracellular replication of L. longbeachae. In addition, the bacterial phagosomes harboring L. longbeachae are enriched with polyubiquitinated proteins in a Dot/Icm system-dependent manner. We further identified 9 L. longbeachae proteins that function as DUBs with distinct ubiquitin chain specificity. Of note, several of the phagosome-associated L. longbeachae DUBs regulate the recruitment of polyubiquitinated proteins to the LCV.

Exploitation of the host exocyst complex by bacterial pathogens.

Intracellular bacterial pathogens remodel the plasma membrane of eukaryotic cells in order to establish infection. A common and well-studied mechanism of plasma membrane remodelling involves bacterial stimulation of polymerization of the host actin cytoskeleton. Here, we discuss recent results showing that several bacterial pathogens also exploit the host vesicular trafficking pathway of 'polarized exocytosis' to expand and reshape specific regions in the plasma membrane during infection. Polarized exocytosis is mediated by an evolutionarily conserved octameric protein complex termed the exocyst. We describe examples in which the bacteria Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Shigella flexneri co-opt the exocyst to promote internalization into human cells or intercellular spread within host tissues. We also discuss results showing that Legionella pneumophila or S. flexneri manipulate exocyst components to modify membrane vacuoles to favour intracellular replication or motility of bacteria. Finally, we propose potential ways that pathogens manipulate exocyst function, discuss how polarized exocytosis might promote infection and highlight the importance of future studies to determine how actin polymerization and polarized exocytosis are coordinated to achieve optimal bacterial infection.

Brucella Egresses from Host Cells Exploiting Multivesicular Bodies.

Host cell egress is a critical step in the life cycle of intracellular pathogens, especially in microbes capable of establishing chronic infections. The Gram-negative bacterium Brucella belongs to such a group of pathogens. Even though much has been done to understand how Brucella avoids killing and multiplies in its intracellular niche, the mechanism that this bacterium deploys to egress from the cell to complete its cycle has been poorly studied. In the manuscript, we quantify the kinetics of bacterial egress and show that Brucella exploits multivesicular bodies to exit host cells. For the first time, we visualized the process of egress in real time by live video microscopy and showed that a population of intracellular bacteria exit from host cells in vacuoles containing multivesicular body-like features. We observed the colocalization of Brucella with two multivesicular markers, namely, CD63 and LBPA, both during the final stages of the intracellular life cycle and in egressed bacteria. Moreover, drugs that either promote or inhibit multivesicular bodies either increased or decreased the number of extracellular bacteria, respectively. Our results strongly suggest that Brucella hijacks multivesicular bodies to exit the host cells to initiate new infection events. IMPORTANCE How intracellular bacterial pathogens egress from host cells has been poorly studied. This is particularly important because this stage of the infectious cycle can have a strong impact on how the host resolves the infection. Brucella is an intracellular pathogen that infects mammals, including humans, and causes a chronic debilitating illness. The bacterium has evolved a plethora of mechanisms to invade host cells, avoid degradation in the endocytic pathway, and actively multiply within a specialized intracellular compartment. However, how this pathogen exits from infected cells to produce reinfection and complete its life cycle is poorly understood. In the manuscript, we shed some light on the mechanisms that are exploited by Brucella to egress from host cells. We observed for the first time the egress of Brucella from infected cells by time-lapse video microscopy, and we found that the bacterium exits in vesicles containing multivesicular bodies (MVBs) features. Moreover, the drug manipulation of MVBs resulted in the alteration of bacterial egress efficiency. Our results indicate that Brucella hijacks MVBs to exit host cells and that this strongly contributes to the reinfection cycle.

Single-cell profiling identifies ACE+ granuloma macrophages as a nonpermissive niche for intracellular bacteria during persistent Salmonella infection.

Macrophages mediate key antimicrobial responses against intracellular bacterial pathogens, such as Salmonella enterica. Yet, they can also act as a permissive niche for these pathogens to persist in infected tissues within granulomas, which are immunological structures composed of macrophages and other immune cells. We apply single-cell transcriptomics to investigate macrophage functional diversity during persistent S. enterica serovar Typhimurium (STm) infection in mice. We identify determinants of macrophage heterogeneity in infected spleens and describe populations of distinct phenotypes, functional programming, and spatial localization. Using an STm mutant with impaired ability to polarize macrophage phenotypes, we find that angiotensin-converting enzyme (ACE) defines a granuloma macrophage population that is nonpermissive for intracellular bacteria, and their abundance anticorrelates with tissue bacterial burden. Disruption of pathogen control by neutralizing TNF is linked to preferential depletion of ACE+ macrophages in infected tissues. Thus, ACE+ macrophages have limited capacity to serve as cellular niche for intracellular bacteria to establish persistent infection.