Coxiella burnetii Pathogenesis: Emphasizing the Role of the Autophagic Pathway.

Coxiella burnetii (C. burnetii), the etiological agent of the Q fever disease, ranks among the most sporadic and persistent global public health concerns. Ruminants are the principal source of human infections and diseases present in both acute and chronic forms. This bacterium is an intracellular pathogen that can survive and reproduce under acidic (pH 4 to 5) and harsh circumstances that contain Coxiella-containing vacuoles. By undermining the autophagy defense system of the host cell, C. burnetii is able to take advantage of the autophagy pathway, which allows it to improve the movement of nutrients and the membrane, thereby extending the vacuole of the reproducing bacteria. For this method to work, it requires the participation of many bacterial effector proteins. In addition, the precise and prompt identification of the causative agent of an acute disease has the potential to delay the onset of its chronic form. Moreover, to make accurate and rapid diagnoses, it is necessary to create diagnostic devices. This review summarizes the most recent research on the epidemiology, pathogenesis, and diagnosis approaches of C. burnetii. This study also explored the complicated relationships between C. burnetii and the autophagic pathway, which are essential for intracellular reproduction and survival in host cells for the infection to be effective.

Intracellular replication of Pseudomonas aeruginosa in epithelial cells requires suppression of the caspase-4 inflammasome.

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with the activation of nuclear factor-κB as measured by p65 relocalization and tumor necrosis factor alpha transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the T3SS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the noncanonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wild-type infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

Interaction between host cell mitochondria and Coxiella burnetii.

In order to successfully establish a replicative niche, intracellular bacterial pathogens must influence eukaryotic cell biology. Vesicle and protein traffic, transcription and translation, metabolism and innate immune signaling are all important elements of the host-pathogen interaction that can be manipulated by intracellular bacterial pathogens. The causative agent of Q fever, Coxiella burnetii, is a mammalian adapted pathogen that replicates in a lysosome-derived pathogen-modified vacuole. C. burnetii establishes this replicative niche by using a cohort of novel proteins, termed effectors, to hijack the mammalian host cell. The functional and biochemical roles of a small number of effectors have been discovered and recent studies have demonstrated that mitochondria are a bona fide target for a subset of these effectors. Various approaches have begun to unravel the role these proteins play at mitochondria during infection, with key mitochondrial functions, including apoptosis and mitochondrial proteostasis, likely influenced by mitochondrially localized effectors. Additionally, mitochondrial proteins likely contribute to the host response to infection. Thus, investigating the interplay between host and pathogen elements at this central organelle will uncover important new understanding of the C. burnetii infection process. With the advent of new technologies and sophisticated omics approaches, we are poised to explore the interaction between host cell mitochondria and C. burnetii with unprecedented spatial and temporal resolution.

Measurement of Salmonella enterica Internalization and Vacuole Lysis in Epithelial Cells.

Establishment of an intracellular niche within mammalian cells is key to the pathogenesis of the gastrointestinal bacterium, Salmonella enterica serovar Typhimurium (S. Typhimurium). Here we will describe how to study the internalization of S. Typhimurium into human epithelial cells using the gentamicin protection assay. The assay takes advantage of the relatively poor penetration of gentamicin into mammalian cells; internalized bacteria are effectively protected from its antibacterial actions. A second assay, the chloroquine (CHQ) resistance assay, can be used to determine the proportion of internalized bacteria that have lysed or damaged their Salmonella-containing vacuole and are therefore residing within the cytosol. Its application to the quantification of cytosolic S. Typhimurium in epithelial cells will also be presented. Together, these protocols provide an inexpensive, rapid, and sensitive quantitative measure of bacterial internalization and vacuole lysis by S. Typhimurium.

The mechanism of chronic intracellular infection with Brucella spp.

Globally, brucellosis is a widespread zoonotic disease. It is prevalent in more than 170 countries and regions. It mostly damages an animal's reproductive system and causes extreme economic losses to the animal husbandry industry. Once inside cells, Brucella resides in a vacuole, designated the BCV, which interacts with components of the endocytic and secretory pathways to ensure bacterial survival. Numerous studies conducted recently have revealed that Brucella's ability to cause a chronic infection depends on how it interacts with the host. This paper describes the immune system, apoptosis, and metabolic control of host cells as part of the mechanism of Brucella survival in host cells. Brucella contributes to both the body's non-specific and specific immunity during chronic infection, and it can aid in its survival by causing the body's immune system to become suppressed. In addition, Brucella regulates apoptosis to avoid being detected by the host immune system. The BvrR/BvrS, VjbR, BlxR, and BPE123 proteins enable Brucella to fine-tune its metabolism while also ensuring its survival and replication and improving its ability to adapt to the intracellular environment.

Legionella pneumophila-mediated host posttranslational modifications.

Legionella pneumophila is a Gram-negative bacterium ubiquitously present in freshwater environments and causes a serious type of pneumonia called Legionnaires' disease. During infections, L. pneumophila releases over 300 effector proteins into host cells through an Icm/Dot type IV secretion system to manipulate the host defense system for survival within the host. Notably, certain effector proteins mediate posttranslational modifications (PTMs), serving as useful approaches exploited by L. pneumophila to modify host proteins. Some effectors catalyze the addition of host protein PTMs, while others mediate the removal of PTMs from host proteins. In this review, we summarize L. pneumophila effector-mediated PTMs of host proteins, including phosphorylation, ubiquitination, glycosylation, AMPylation, phosphocholination, methylation, and ADP-ribosylation, as well as dephosphorylation, deubiquitination, deAMPylation, deADP-ribosylation, dephosphocholination, and delipidation. We describe their molecular mechanisms and biological functions in the regulation of bacterial growth and Legionella-containing vacuole biosynthesis and in the disruption of host immune and defense machinery.

Identification of Type 4B Secretion System Substrates That Are Conserved among Coxiella burnetii Genomes and Promote Intracellular Growth.

Coxiella burnetii is a Gram-negative pathogen that infects a variety of mammalian hosts. Infection of domesticated ewes can cause fetal abortion, whereas acute human infection normally manifests as the flu-like illness Q fever. Successful host infection requires replication of the pathogen within the lysosomal Coxiella-containing vacuole (CCV). The bacterium encodes a type 4B secretion system (T4BSS) that delivers effector proteins into the host cell. Disruption of C. burnetii T4BSS effector export abrogates CCV biogenesis and bacterial replication. Over 150 C. burnetii T4BSS substrates have been designated often based on heterologous protein translocation by the Legionella pneumophila T4BSS. Cross-genome comparisons predict that many of these T4BSS substrates are truncated or absent in the acute-disease reference strain C. burnetii Nine Mile. This study investigated the function of 32 proteins conserved among diverse C. burnetii genomes that are reported to be T4BSS substrates. Despite being previously designated T4BSS substrates, many of the proteins were not translocated by C. burnetii when expressed fused to the CyaA or BlaM reporter tags. CRISPR interference (CRISPRi) indicated that of the validated C. burnetii T4BSS substrates, CBU0122, CBU1752, CBU1825, and CBU2007 promote C. burnetii replication in THP-1 cells and CCV biogenesis in Vero cells. When expressed in HeLa cells tagged at its C or N terminus with mCherry, CBU0122 localized to the CCV membrane and the mitochondria, respectively. Collectively, these data further define the repertoire of bona fide C. burnetii T4BSS substrates. IMPORTANCE Coxiella burnetii secretes effector proteins via a T4BSS that are required for successful infection. Over 150 C. burnetii proteins are reported to be T4BSS substrates and often by default considered putative effectors, but few have assigned functions. Many C. burnetii proteins were designated T4BSS substrates using heterologous secretion assays in L. pneumophila and/or have coding sequences that are absent or pseudogenized in clinically relevant C. burnetii strains. This study examined 32 previously reported T4BSS substrates that are conserved among C. burnetii genomes. Of the proteins tested that were previously designated T4BSS substrates using L. pneumophila, most were not exported by C. burnetii. Several T4BSS substrates that were validated in C. burnetii also promoted pathogen intracellular replication and one trafficked to late endosomes and the mitochondria in a manner suggestive of effector activity. This study identified several bona fide C. burnetii T4BSS substrates and further refined the methodological criteria for their designation.

Syntaxin 3 SPI-2 dependent crosstalk facilitates the division of Salmonella containing vacuole.

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Reports also suggest that Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. Salmonella containing vacuole (SCV) harbours recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis remains unclear. Upon knockdown of STX3, we observed a reduction in bacterial proliferation, which is concomitantly restored upon the overexpression of STX3. Live-cell imaging of Salmonella-infected cells showed that STX3 localises to the SCV membranes and thus might help in the fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found the interaction STX3-SCV was abrogated when we infected with SPI-2 encoded Type 3 secretion system (T3SS) apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS apparatus mutant (STM ∆invC). These observations were also consistent in the mice model of Salmonella infection. Together, these results shed light on the effector molecules secreted through T3SS encoded by SPI-2, possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and help to maintain a single bacterium per vacuole.

Eat or Be Eaten: Strategies Used by Legionella to Acquire Host-Derived Nutrients and Evade Lysosomal Degradation.

To replicate within host cells, bacterial pathogens must acquire host-derived nutrients while avoiding degradative antimicrobial pathways. Fundamental insights into bacterial pathogenicity have been revealed by bacteria of the genus Legionella, which naturally parasitize free-living protozoa by establishing a membrane-bound replicative niche termed the Legionella-containing vacuole (LCV). Biogenesis of the LCV and intracellular replication rely on rapid evasion of the endocytic pathway and acquisition of host-derived nutrients, much of which is mediated by bacterial effector proteins translocated into host cells by a Dot/Icm type IV secretion system. Billions of years of co-evolution with eukaryotic hosts and broad host tropism have resulted in expansion of the Legionella genome to accommodate a massive repertoire of effector proteins that promote LCV biogenesis, safeguard the LCV from endolysosomal maturation, and mediate the acquisition of host nutrients. This minireview is focused on the mechanisms by which an ancient intracellular pathogen leverages effector proteins and hijacks host cell biology to obtain essential host-derived nutrients and prevent lysosomal degradation.

MicroRNAs Contribute to Host Response to Coxiella burnetii.

MicroRNAs (miRNAs), a class of small noncoding RNAs, are critical to gene regulation in eukaryotes. They are involved in modulating a variety of physiological processes, including the host response to intracellular infections. Little is known about miRNA functions during infection by Coxiella burnetii, the causative agent of human Q fever. This bacterial pathogen establishes a large replicative vacuole within macrophages by manipulating host processes such as apoptosis and autophagy. We investigated miRNA expression in C. burnetii-infected macrophages and identified several miRNAs that were down- or upregulated during infection. We further explored the functions of miR-143-3p, an miRNA whose expression is downregulated in macrophages infected with C. burnetii, and show that increasing the abundance of this miRNA in human cells results in increased apoptosis and reduced autophagy-conditions that are unfavorable to C. burnetii intracellular growth. In sum, this study demonstrates that C. burnetii infection elicits a robust miRNA-based host response, and because miR-143-3p promotes apoptosis and inhibits autophagy, downregulation of miR-143-3p expression during C. burnetii infection likely benefits the pathogen.

Lysosomal trafficking regulator restricts intracellular growth of Coxiella burnetii by inhibiting the expansion of Coxiella-containing vacuole and upregulating nos2 expression.

Coxiella burnetii is an obligate intracellular bacterium that causes Q fever, a zoonotic disease typically manifests as a severe flu-illness. After invading into the host cells, C. burnetii delivers effectors to regulate the vesicle trafficking and fusion events to form a large and mature Coxiella-containing vacuole (CCV), providing sufficient space and nutrition for its intracellular growth and proliferation. Lysosomal trafficking regulator (LYST) is a member of the Beige and Chediak-Higashi syndrome (BEACH) family, which regulates the transport of vesicles to lysosomes and regulates TLR signaling pathway, but the effect of LYST on C. burnetii infection is unclear. In this study, a series of experiments has been conducted to investigate the influence of LYST on intracellular growth of C. burnetii. Our results showed that lyst transcription was up-regulated in the host cells after C. burnetii infection, but there is no significant change in lyst expression level after infection with the Dot/Icm type IV secretion system (T4SS) mutant strain, while CCVs expansion and significantly increasing load of C. burnetii appeared in the host cells with a silenced lyst gene, suggesting LYST inhibits the intracellular proliferation of C. burnetii by reducing CCVs size. Then, the size of CCVs and the load of C. burnetii in the HeLa cells pretreated with E-64d were significantly decreased. In addition, the level of iNOS was decreased significantly in LYST knockout THP-1 cells, which was conducive to the intracellular replication of C. burnetii. This data is consistent with the phenotype of L-NMMA-treated THP-1 cells infected with C. burnetii. Our results revealed that the upregulation of lyst transcription after infection is due to effector secretion of C. burnetii and LYST inhibit the intracellular replication of C. burnetii by reducing the size of CCVs and inducing nos2 expression.

5' Untranslated mRNA Regions Allow Bypass of Host Cell Translation Inhibition by Legionella pneumophila.

Legionella pneumophila grows within membrane-bound vacuoles in alveolar macrophages during human disease. Pathogen manipulation of the host cell is driven by bacterial proteins translocated through a type IV secretion system (T4SS). Although host protein synthesis during infection is arrested by the action of several of these translocated effectors, translation of a subset of host proteins predicted to restrict the pathogen is maintained. To identify the spectrum of host proteins selectively synthesized after L. pneumophila challenge, macrophages infected with the pathogen were allowed to incorporate the amino acid analog azidohomoalanine (AHA) during a 2-h time window, and newly synthesized macrophage proteins were isolated by orthogonal chemistry followed by mass spectrometry. Among the proteins isolated were interferon-stimulated genes as well as proteins translated from highly abundant transcripts. Surprisingly, a large number of the identified proteins were from low-abundance transcripts. These proteins were predicted to be among the most efficiently translated per unit transcript in the cell based on ribosome profiling data sets. To determine if high ribosome loading was a consequence of efficient translation initiation, the 5' untranslated regions (5' UTR) of transcripts having the highest and lowest predicted loading levels were inserted upstream of a reporter, and translation efficiency was determined in response to L. pneumophila challenge. The efficiency of reporter expression largely correlated with predicted ribosome loading and lack of secondary structure. Therefore, determinants in the 5' UTR allow selected host cell transcripts to overcome a pathogen-driven translation blockade.

Developmental Transitions Coordinate Assembly of the Coxiella burnetii Dot/Icm Type IV Secretion System.

Coxiella burnetii is an obligate intracellular bacterial pathogen that has evolved a unique biphasic developmental cycle. The infectious form of C. burnetii is the dormant small cell variant (SCV), which transitions to a metabolically active large cell variant (LCV) that replicates inside the lysosome-derived host vacuole. A Dot/Icm type IV secretion system (T4SS), which can deliver over 100 effector proteins to host cells, is essential for the biogenesis of the vacuole and intracellular replication. How the distinct C. burnetii life cycle impacts the assembly and function of the Dot/Icm T4SS has remained unknown. Here, we combine advanced cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) imaging to visualize all developmental transitions and the assembly of the Dot/Icm T4SS in situ. Importantly, assembled Dot/Icm machines were not present in the infectious SCV. The appearance of the assembled Dot/Icm machine correlated with the transition of the SCV to the LCV intracellularly. Furthermore, temporal characterization of C. burnetii morphological changes revealed regions of the inner membrane that invaginate to form tightly packed stacks during the LCV-to-SCV transition at late stages of infection, which may enable the SCV-to-LCV transition that occurs upon infection of a new host cell. Overall, these data establish how C. burnetii developmental transitions control critical bacterial processes to promote intracellular replication and transmission.

Automated Analysis of Intracellular Phenotypes of Salmonella using ImageJ.

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96-well plates. Imaging was performed using an automated fluorescence microscope. Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for high-throughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Coxiella burnetii Plasmid Effector B Promotes LC3-II Accumulation and Contributes To Bacterial Virulence in a SCID Mouse Model.

Coxiella burnetii, the causative agent of zoonotic Q fever, is characterized by replicating inside the lysosome-derived Coxiella-containing vacuole (CCV) in host cells. Some effector proteins secreted by C. burnetii have been reported to be involved in the manipulation of autophagy to facilitate the development of CCVs and bacterial replication. Here, we found that the Coxiella plasmid effector B (CpeB) localizes on vacuole membrane targeted by LC3 and LAMP1 and promotes LC3-II accumulation. Meanwhile, the C. burnetii strain lacking the QpH1 plasmid induced less LC3-II accumulation, which was accompanied by smaller CCVs and lower bacterial loads in THP-1 cells. Expression of CpeB in the strain lacking QpH1 led to restoration in LC3-II accumulation but had no effect on the smaller CCV phenotype. In the severe combined immune deficiency (SCID) mouse model, infections with the strain expressing CpeB led to significantly higher bacterial burdens in the spleen and liver than its parent strain devoid of QpH1. We also found that CpeB targets Rab11a to promote LC3-II accumulation. Intratracheally inoculated C. burnetii resulted in lower bacterial burdens and milder lung lesions in Rab11a conditional knockout (Rab11a-/- CKO) mice. Collectively, these results suggest that CpeB promotes C. burnetii virulence by inducing LC3-II accumulation via a pathway involving Rab11a.

Adaptations of intracellular bacteria to vacuolar or cytosolic niches.

Invasive bacteria colonise their host tissues by establishing niches inside eukaryotic cells, where they grow either in the cytosol or inside a specialised vacuole. These two distinct intracellular lifestyles both present benefits but also impose various constraints on pathogenic microorganisms, in terms of nutrient acquisition, space requirements, exposure to immune responses, and ability to disseminate. Here we review the major characteristics of cytosolic and vacuolar lifestyles and the strategies used by bacteria to overcome challenges specific to each compartment. Recent research providing evidence that these scenarios are not mutually exclusive is presented, with the dual lifestyles of two foodborne pathogens, Listeria monocytogenes and Salmonella Typhimurium, discussed in detail. Finally, we elaborate on the conceptual implications of polyvalence from the perspective of host-pathogen interactions.

Legionella pneumophila modulates host energy metabolism by ADP-ribosylation of ADP/ATP translocases.

The intracellular pathogen Legionella pneumophila delivers more than 330 effectors into host cells by its Dot/Icm secretion system. Those effectors direct the biogenesis of the Legionella-containing vacuole (LCV) that permits its intracellular survival and replication. It has long been documented that the LCV is associated with mitochondria and a number of Dot/Icm effectors have been shown to target to this organelle. Yet, the biochemical function and host cell target of most of these effectors remain unknown. Here, we found that the Dot/Icm substrate Ceg3 (Lpg0080) is a mono-ADP-ribosyltransferase that localizes to the mitochondria in host cells where it attacks ADP/ATP translocases by ADP-ribosylation, and blunts their ADP/ATP exchange activity. The modification occurs on the second arginine residue in the -RRRMMM- element, which is conserved among all known ADP/ATP carriers from different organisms. Our results reveal modulation of host energy metabolism as a virulence mechanism for L. pneumophila.

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

The role of membrane contact sites at the bacteria-host interface.

Membrane contact sites (MCSs) refer to the areas of close proximity between heterologous membranes. A growing body of evidence indicates that MCSs are involved in important cellular functions, such as cellular material transfer, organelle biogenesis, and cell growth. Importantly, the study of MCSs at the bacteria-host interface is an emerging popular research topic. Intracellular bacterial pathogens have evolved a variety of fascinating strategies to interfere with MCSs by injecting effectors into infected host cells. Bacteria-containing vacuoles establish direct physical contact with organelles within the host, ensuring vacuolar membrane integrity and energy supply from host organelles and protecting the vacuoles from the host endocytic pathway and lysosomal degradation. An increasing number of bacterial effectors from various bacterial pathogens hijack components of host MCSs to form the vacuole-organelle MCSs for material exchange. MCS-related events have been identified as new mechanisms of microbial pathogenesis to greatly improve bacterial survival and replication within host cells. In this review, we will discuss the recent advances in MCSs at the bacteria-host interface, focussing on the roles of MCSs mediated by bacterial effectors in microbial pathogenesis.

A Role for Taok2 in Listeria monocytogenes Vacuolar Escape.

The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.