Intracellular Pathogens Research Articles

Apicomplexan Espionage: Orchestrated Miscommunication at the Host-Parasite Interface.

Intracellular parasites, including Toxoplasma and Plasmodium, are entirely reliant on the active scavenging of host-derived nutrients to fuel their replicative cycle, as they are confined within a specialized membrane-bound compartment, the parasitophorous vacuole (PV). Initial observations, based on the proximity of host vesicles to the parasitophorous vacuole membrane (PVM), suggested that parasites utilize host vesicles to obtain essential nutrients. However, mounting evidence has now unequivocally demonstrated that intracellular pathogens establish membrane contacts with host organelles, establishing control over host cellular machinery. These intimate interactions enable the parasites to gain unimpeded access to cytosolic resources critical for development while evading host immune responses. This review consolidates the latest advancements in understanding the molecular machinery driving these transkingdom contacts and their functional roles. Further investigation into these processes promises to revolutionize our understanding of organelle communication, with profound implications for identifying new therapeutic targets and strategies.

Deletion of glycosyltransferase galE impairs the InlB anchoring and pathogenicity of Listeria monocytogenes

ABSTRACT Listeria monocytogenes (L. monocytogenes) is a foodborne intracellular pathogen that causes serious disease in both humans and animals. InlB is the major internalin protein of L. monocytogenes, which anchors to the bacterial surface and mediates its invasion into various host cells. Recent studies have shown that galactosylation of the cell wall polymer wall teichoic acid (WTA) is essential for InlB anchoring on the cell surface of L. monocytogenes serotype 4b strains. Galactosylation of WTA is exerted by the coordinated action of several glycosyltransferases, including GalU, GalE, GtcA, GttA, and GttB. Among these glycosyltransferases, GttA and GttB are specific to serotype 4b strains, whereas GalE, GalU, and GtcA are conserved across all serotypes. The role of GalE in InlB anchoring and L. monocytogenes pathogenicity remains unclear. In this study, we deleted the galE gene, which is involved in galactosylation, from L. monocytogenes strain ScottA. We found that galE deletion reduced InlB anchoring, weakened bacterial adhesion and invasion of Caco-2 cells (human colorectal adenocarcinoma cells) and MGC803 cells (human gastric carcinoma cells), increased phagocytosis but decreased proliferation in RAW264.7 cells (mouse mononuclear macrophage leukaemia cells), and decreased bacteria load, mortality, and tissue damage in infected mice. Taken together, galE deletion significantly reduced the anchoring of InlB and weakened the pathogenicity of L. monocytogenes. This finding provides new insights into the correlation between cell wall modification and pathogenicity of L. monocytogenes.

Epigenetic Mechanisms Induced by Mycobacterium tuberculosis to Promote Its Survival in the Host

Tuberculosis caused by the obligate intracellular pathogen, Mycobacterium tuberculosis, is one among the prime causes of death worldwide. An urgent remedy against tuberculosis is of paramount importance in the current scenario. However, the complex nature of this appalling disease contributes to the limitations of existing medications. The quest for better treatment approaches is driving the research in the field of host epigenomics forward in context with tuberculosis. The interplay between various host epigenetic factors and the pathogen is under investigation. A comprehensive understanding of how Mycobacterium tuberculosis orchestrates such epigenetic factors and favors its survival within the host is in increasing demand. The modifications beneficial to the pathogen are reversible and possess the potential to be better targets for various therapeutic approaches. The mechanisms, including histone modifications, DNA methylation, and miRNA modification, are being explored for their impact on pathogenesis. In this article, we are deciphering the role of mycobacterial epigenetic regulators on various strategies like cytokine expression, macrophage polarization, autophagy, and apoptosis, along with a glimpse of the potential of host-directed therapies.

A natural killer cell mimic against intracellular pathogen infections.

In the competition between the pathogen infection and the host defense, infectious microorganisms may enter the host cells by evading host defense mechanisms and use the intracellular biomolecules as replication nutrient. Among them, intracellular Staphylococcus aureus relies on the host cells to protect itself from the attacks by antibiotics or immune system to achieve long-term colonization in the host, and the consequent clinical therapeutic failures and relapses after antibiotic treatment. Here, we demonstrate that intracellular S. aureus surviving well even in the presence of vancomycin can be effectively eliminated using an emerging cell-mimicking therapeutic strategy. These cell mimics with natural killer cell-like activity (NKMs) are composed of a redox-responsive degradable carrier, and perforin and granzyme B within the carrier. NKMs perform far more effectivly than clinical antibiotics in treating intracellular bacterial infections, providing a direct evidence of the NK cell-mimicking immune mechanism in the treatment of intracellular S. aureus.

Bartonella Species in Small Mammals in Turkey: Bartonella bilalgolemii sp. nov. Isolated from a Ural Field Mouse (Apodemus uralensis).

Background: The genus Bartonella is composed of Gram-negative, fastidious, facultative intracellular bacteria that can cause bacteremia in mammals and various disorders in humans. Rodents have been reported as reservoirs of more than 30 Bartonella species, seven of which cause zoonotic infections. Materials and Methods: In the present study, the isolation of Bartonella sp. was attempted from 150 spleen samples from 13 rodent species (mostly Apodemus species) from three geographically different regions in Turkey. Results: Bartonella sp. was successfully isolated from 65 of these 150 samples (43%). The prevalences of Bartonella sp. in tested rodents in the regions of Giresun, Yozgat, and Burdur were 68%, 44%, and 16%, respectively. Using polymerase chain reaction/sequence analysis of the citrate synthase-coding gene (gltA), Bartonellaisolates were classified seven species including B. taylorii, B. grahamii, B. birtlesii, B. mastomydis, and three putatively new Bartonella species. We performed further identification techniques for one of the three Bartonella species that were different from the validated Bartonella species according to the gltA sequence analysis. Conclusion: Here, we report the genomic and phenotypic characterization of Bartonella sp. strain G70 that was isolated from the splenic tissue of an Apodemus uralensis (Pallas 1881), the Ural field mouse, captured in the Giresun region of northeastern Turkey. Bartonella sp. strainG70 (RSKK 22001) was characterized by whole genome and partial gene (gltA, 16S ribosomal RNA) sequencing and comparison, scanning electron microscopy, biochemical tests, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This novel Bartonella is a Gram-negative, rod-shaped bacterium and has neither flagella nor pili. The genome from strain G70 was 1,606,969-bp-long with a G + C content of 35.7%. Bartonella rochalimae was found to be the closest phylogenetic relative of strain G70 (OrthoANI = 90.5%, digital DNA-DNA hybridization = 41.4%). We therefore propose that this new species be named Bartonella bilalgolemii sp. nov. with strain G70T as the type strain.

Leishmania protein KMP-11 modulates cholesterol transport and membrane fluidity to facilitate host cell invasion.

The first step of successful infection by any intracellular pathogen relies on its ability to invade its host cell membrane. However, the detailed structural and molecular understanding underlying lipid membrane modification during pathogenic invasion remains unclear. In this study, we show that a specific Leishmania donovani (LD) protein, KMP-11, forms oligomers that bridge LD and host macrophage (MΦ) membranes. This KMP-11 induced interaction between LD and MΦ depends on the variations in cholesterol (CHOL) and ergosterol (ERG) contents in their respective membranes. These variations are crucial for the subsequent steps of invasion, including (a) the initial attachment, (b) CHOL transport from MΦ to LD, and (c) detachment of LD from the initial point of contact through a liquid ordered (Lo) to liquid disordered (Ld) membrane-phase transition. To validate the importance of KMP-11, we generate KMP-11 depleted LD, which failed to attach and invade host MΦ. Through tryptophan-scanning mutagenesis and synthesized peptides, we develop a generalized mathematical model, which demonstrates that the hydrophobic moment and the symmetry sequence code at the membrane interacting protein domain are key factors in facilitating the membrane phase transition and, consequently, the host cell infection process by Leishmania parasites.

Seroprevalence of Toxoplasma gondii in Cattle in Southern Egypt: Do Milk and Serum Samples Tell the Same Story?

Toxoplasma gondii is an intracellular protozoan parasite of veterinary and public health importance. Infection may lead to abortion in susceptible pregnant animals and women, and potentially fatal health complications in immunocompromised individuals. In this study, we aimed to provide an update on the seroprevalence of, and risk factors for, T. gondii antibodies in cattle from Qena, southern Egypt. Additionally, we investigated if raw milk and serum samples from the same animals reacted similarly in a commercial ELISA, thus potentially reducing the invasiveness of future serosurveillance studies. Cattle serum samples (n = 362) from three locations in the Qena governorate (Qena, Qus, and Al Waqf cities), of both sexes and different ages were collected. From most dairy cows, a corresponding milk sample (n = 154) was additionally obtained. We found that the overall seroprevalence in serum samples was 9.1% (33/362). Increasing age was the sole risk factor identified in our study among all tested parameters (location, age, sex, lactating yes or no). Thus, older cattle (more than 3 years old) exhibited significantly higher rates of T. gondii antibodies (11.7%; p = 0.033, odd ratio = 4.3) in comparison to animals younger than 1 year (2.9%). In the corresponding serum and milk samples, the prevalence was 12.3% (19/154) in serum samples, and 9.7% (15/154) in milk samples, respectively. A high correlation was observed between the two sample types with a concordance of 97.4%, a kappa value of 0.87, and a Pearson r correlation coefficient of 0.85. When the serum ELISA was taken as the gold standard, the milk ELISA had the following characteristics: sensitivity (78.9%), specificity (100%), positive predictive value (100%), negative predictive value (97.1%), and area under the receiver operating characteristic (ROC) curve (0.6, p = 0.0011). In this study, we confirmed the frequent occurrence of T. gondii antibodies in cattle in southern Egypt and demonstrated that non-invasive milk samples may be used instead of serum samples for seroprevalence studies in dairy cows.

Evolution shapes and conserves genomic signatures in viruses

The genomic signature of an organism captures the characteristics of repeated oligonucleotide patterns in its genome 1, such as oligomer frequencies, GC content, and differences in codon usage. Viruses, however, are obligate intracellular parasites that are dependent on their host cells for replication, and information about genomic signatures in viruses has hitherto been sparse.Here, we investigate the presence and specificity of genomic signatures in 2,768 eukaryotic viral species from 105 viral families, aiming to illuminate dependencies and selective pressures in viral genome evolution. We demonstrate that most viruses have highly specific genomic signatures that often also differ significantly between species within the same family. The species-specificity is most prominent among dsDNA viruses and viruses with large genomes. We also reveal consistent dissimilarities between viral genomic signatures and those of their host cells, although some viruses present slight similarities, which may be explained by genetic adaptation to their native hosts. Our results suggest that significant evolutionary selection pressures act upon viral genomes to shape and preserve their genomic signatures, which may have implications for the field of synthetic biology in the construction of live attenuated vaccines and viral vectors.

Mitochondrial Dysfunction and Metabolic Disturbances Induced by Viral Infections

Viruses are intracellular parasites that utilize organelles, signaling pathways, and the bioenergetics machinery of the cell to replicate the genome and synthesize proteins to build up new viral particles. Mitochondria are key to supporting the virus life cycle by sustaining energy production, metabolism, and synthesis of macromolecules. Mitochondria also contribute to the antiviral innate immune response. Here, we describe the different mechanisms involved in virus–mitochondria interactions. We analyze the effects of viral infections on the metabolism of glucose in the Warburg phenotype, glutamine, and fatty acids. We also describe how viruses directly regulate mitochondrial function through modulation of the activity of the electron transport chain, the generation of reactive oxygen species, the balance between fission and fusion, and the regulation of voltage-dependent anion channels. In addition, we discuss the evasion strategies used to avoid mitochondrial-associated mechanisms that inhibit viral replication. Overall, this review aims to provide a comprehensive view of how viruses modulate mitochondrial function to maintain their replicative capabilities.

Distinct impacts of each anti-anti-sigma factor ortholog of the chlamydial Rsb partner switching mechanism on development in Chlamydia trachomatis.

Partner switching mechanisms (PSMs) are signal transduction systems comprised of a sensor phosphatase (RsbU), an anti-sigma factor (RsbW, kinase), an anti-anti-sigma factor (RsbV, the RsbW substrate), and a target sigma factor. Chlamydia spp. are obligate intracellular bacterial pathogens of animals that undergo a developmental cycle transitioning between the infectious elementary body (EB) and replicative reticulate body (RB) within a host cell-derived vacuole (inclusion). Secondary differentiation events (RB to EB) are transcriptionally regulated, in part, by the housekeeping sigma factor (σ66) and two late-gene sigma factors (σ54 and σ28). Prior research supports that the PSM in Chlamydia trachomatis regulates availability of σ66. Pan-genome analysis revealed that PSM components are conserved across the phylum Chlamydiota, with Chlamydia spp. possessing an atypical arrangement of two anti-anti-sigma factors, RsbV1 and RsbV2. Bioinformatic analyses support RsbV2 as the homolog to the pan-genome-conserved RsbV with RsbV1 as an outlier. This, combined with in vitro data, indicates that RsbV1 and RsbV2 are structurally and biochemically distinct. Reduced levels or overexpression of RsbV1/RsbV2 did not significantly impact C. trachomatis growth or development. In contrast, overexpression of a non-phosphorylatable RsbV2 S55A mutant, but not overexpression of an RsbV1 S56A mutant, resulted in a 3 log reduction in infectious EB production without reduction in genomic DNA (total bacteria) or inclusion size, suggesting a block in secondary differentiation. The block was corroborated by reduced production of σ54/28-regulated late proteins and via transmission electron microscopy.IMPORTANCEChlamydia trachomatis is the leading cause of reportable bacterial sexually transmitted infections (STIs) and causes the eye infection trachoma, a neglected tropical disease. Broad-spectrum antibiotics used for treatment can lead to microbiome dysbiosis and increased antibiotic resistance development in other bacteria, and treatment failure for chlamydial STIs is a recognized clinical problem. Here, we show that disruption of a partner switching mechanism (PSM) significantly reduces infectious progeny production via blockage of reticulate body to elementary body differentiation. We also reveal a novel PSM expansion largely restricted to the species infecting animals, suggesting a role in pathogen evolution. Collectively, our results highlight the chlamydial PSM as a key regulator of development that could be a potential target for novel therapeutics.

The arginine/ornithine binding protein ArgT plays an essential role in Brucella neotomae/Brucella melitensis to prevent intracellular killing and contribute to chronic persistence in the host

ABSTRACT Brucella species are facultative intracellular bacterial pathogens that cause the contagious zoonotic disease, brucellosis. Brucella spp. infect a wide range of animals, including livestock, wild animals, and marine mammals. Compared with other invasive bacterial pathogens, partial information is available on the virulence factors of Brucella that enable them to survive in the host. Here, we performed transposon-based random mutagenesis of B. neotomae and identified the arginine/ornithine binding protein, ArgT, as one of the crucial virulence determinants of Brucella. Deleting ArgT from B. neotomae or B. melitensis resulted in its attenuation in macrophages, which was restored upon complementation with an ArgT expression plasmid. We observed that macrophages infected with ΔArgT-B. neotomae produced elevated levels of NO due to the inability of these mutants to deplete the host intracellular arginine through their importer. Furthermore, defective survival of ΔArgT B. neotomae and B. melitensis was observed in the infected mice, which correlated with enhanced NO production in the mice. Our studies revealed that ArgT plays a vital role in preventing intracellular killing and contributes to the chronic persistence of B. neotomae/B. melitensis in the host. This study highlights the essential role of arginine in clearing intracellular infections and the subversion of this host defence mechanism by intracellular pathogens for their chronic persistence.

Specialized contact sites regulate the fusion of chlamydial inclusion membranes

The intracellular bacterial pathogen Chlamydia trachomatis replicates within a membrane-bound compartment called the inclusion. Upon infection with several chlamydiae, each bacterium creates its own inclusion, resulting in multiple inclusions within each host cell. Ultimately, these inclusions fuse together in a process that requires the chlamydial protein IncA. Here, we show that inclusions form unique contact sites (inclusion contact sites, ICSs) prior to fusion, that serve as fusogenic platforms in which specific lipids and chlamydial proteins concentrate. Fusion depends on IncA clustering within ICSs and is regulated by PI(3,4)P2 and sphingolipids. As IncA concentrates within ICSs, its C-terminus likely interacts in trans with IncA on the apposing membrane, securing a high concentration of IncA at fusion sites. This regulatory mechanism contrasts with eukaryotic or viral fusion systems that are either composed of multiple proteins or use a change in pH to initiate membrane fusion. Thus, our study demonstrates that Chlamydia-mediated membrane fusion is primarily regulated by specific structural domains in IncA and its local organization on the inclusion membrane, which is affected by the host cell lipid composition.

Genetic diversity of Bartonella species in small mammals in the Qinghai Menyuan section of Qilian Mountain National Park, China

Bartonella are vector-borne gram-negative facultative intracellular bacteria that can infect a wide spectrum of mammals, and are recognized as emerging human pathogens. This study aimed to investigate the prevalence and molecular characteristics of Bartonella infections in small mammals within the Qinghai Menyuan section of Qilian Mountain National Park, China. Small mammals were captured, and the liver, spleen and kidney were collected for Bartonella detection and identification using a combination of real-time PCR targeting the transfer-mRNA (ssrA) gene and followed by sequencing of the citrate synthase (gltA) gene. A total of 52 rodents were captured, and 36 rodents were positive for Bartonella, with a positivity rate of 69.23% (36/52). Bartonella was detected in Cricetulus longicaudatus, Microtus oeconomus, Mus musculus, and Ochotona cansus. The positivity rate was significantly different in the different habitats. Two Bartonella species were observed, including Bartonella grahamii and Bartonella heixiaziensis, and B. grahamii was the dominant epidemic strain in this area. Phylogenetic analysis showed that B. grahamii mainly clustered into two clusters, which were closely related to the Apodemus isolates from China and Japan and the local plateau pika isolates, respectively. In addition, genetic polymorphism analysis showed that B. grahamii had high genetic diversity, and its haplotype had certain regional differences and host specificity. Overall, high prevalence of Bartonella in small mammals in the Qinghai Menyuan section of Qilian Mountain National Park. B. grahamii is the dominant strain with high genetic diversity and potential pathogenicity to humans, and corresponding control measures should be considered.

FtsK Initiates the Assembly of a Unique Divisome Complex in the FtsZ-less Chlamydia trachomatis.

Chlamydia trachomatis serovar L2 ( Ct ), an obligate intracellular bacterium that does not encode FtsZ, divides by a polarized budding process. In the absence of FtsZ, we show that divisome assembly in Ct is initiated by FtsK, a chromosomal translocase. Chlamydial FtsK forms discrete foci at the septum and at the base of the progenitor mother cell, and our data indicate that FtsK foci at the base of the mother cell mark the location of nascent divisome complexes that form at the site where a daughter cell will emerge in the next round of division. The divisome in Ct has a hybrid composition, containing elements of the divisome and elongasome from other bacteria, and FtsK is recruited to nascent divisomes prior to the other chlamydial divisome proteins assayed, including the PBP2 and PBP3 transpeptidases, and MreB and MreC. Knocking down FtsK prevents divisome assembly in Ct and inhibits cell division and septal peptidoglycan synthesis. We further show that MreB does not function like FtsZ and serve as a scaffold for the assembly of the Ct divisome. Rather, MreB is one of the last proteins recruited to the chlamydial divisome, and it is necessary for the formation of septal peptidoglycan rings. Our studies illustrate the critical function of chlamydial FtsK in coordinating divisome assembly and peptidoglycan synthesis in this obligate intracellular bacterial pathogen.

Dissecting the role of transcription factor AP2-M in Babesia asexual replication.

Babesia spp. are obligate intracellular parasites that invade host cells to complete their asexual development and transmission. Here, we identified a transcription factor AP2-M (BXIN_0799) in Babesia sp. Xinjiang (Bxj), a member of the Apicomplexan AP2 family, which regulates gene expression related to red blood cell (RBC) invasion and cell cycle progression. Our genome-wide analysis of (Cut-Tag) data shows that AP2-M specifically recognized DNA motifs in the promoters of target genes. AP2-M target genes included other AP2 gene family members and epigenetic markers, which could modulate gene expression involved in RBC invasion, merozoite morphology, and cell cycle phases, as indicated by RNA sequencing, proteomics, and single-cell RNA sequencing (scRNA-seq) data from an ap2-m gene disrupted strain (AP2-M (-)). We conclude that AP2-M appeared to contribute to the process of red blood cell invasion, maintain merozoite morphology, and cell cycle progression through GS and MS phases.

Type 1 diabetes and parasite infection: An exploratory study in NOD mice.

Microorganisms have long been suspected to influence the outcome of immune-related syndromes, particularly autoimmune diseases. Type 1 diabetes (T1D) results from the autoimmune destruction of the insulin-producing beta cells of pancreatic islets, causing high glycemia levels. Genetics is part of its aetiology, but environmental factors, particularly infectious microorganisms, also play a role. Bacteria, viruses, and parasites influence the outcome of T1D in mice and humans. We used nonobese diabetic (NOD) mice, which spontaneously develop T1D, to investigate the influence of a parasitic infection, leishmaniasis. Leishmania amazonensis is an intracellular eukaryotic parasite that replicates predominantly in macrophages and is responsible for cutaneous leishmaniasis. The implication of Th1 immune responses in T1D and leishmaniasis led us to study this parasite in the NOD mouse model. We previously constructed osteopontin knockout mice with a NOD genetic background and demonstrated that this protein plays a role in the T1D phenotype. In addition, osteopontin (OPN) has been found to play a role in the immune response to various infectious microorganisms and to be implicated in other autoimmune conditions, such as multiple sclerosis in humans and experimental autoimmune encephalomyelitis (EAE) in mice. We present herein data demonstrating the role of OPN in the response to Leishmania in NOD mice and the influence of this parasitic infection on T1D. This exploratory study aimed to investigate the environmental infectious component of the autoimmune response, including Th1 immunity, which is common to both T1D and leishmaniasis.

Toxoplasma gondii infection induces the expression of the chemokine CXCL16 in macrophages to promote chemoattraction of CXCR6+ cells.

CXCL16 is a multifaceted chemokine expressed by macrophages and other immune cells in response to viral and bacterial pathogens. However, few studies have investigated its role in parasitic infections. The obligate intracellular parasite Toxoplasma gondii (T. gondii) is the causative agent of toxoplasmosis, an infection with potentially deleterious consequences in immunocompromised individuals and the developing fetus of acutely infected pregnant women. Chemokines are critical mediators of host defense and, as such, dysregulation of their expression is a subversion strategy often employed by the parasite to ensure its survival. Herein, we report that types I and II T. gondii strains upregulated the expression of both transmembrane and soluble forms of CXCL16 in infected bone marrow-derived macrophages (BMDM). Exposure to soluble T. gondii antigens (STAg) and to excreted-secreted proteins (TgESP) led to the induction of CXCL16. Cxcl16 mRNA abundance and CXCL16 protein levels increased in a time-dependent manner upon T. gondii infection. Importantly, conditioned medium (CM) collected from T. gondii-infected wild-type (WT) macrophage cultures promoted the migration of RAW264.7 cells expressing CXCR6, the cognate receptor of CXCL16, an effect that was significantly reduced by a neutralizing anti-CXCL16 antibody or use of CM from CXCL16 knockout (KO) macrophages. Lastly, T. gondii-driven CXCL16 expression appeared to modulate cytokine-induced (IL-4 + IL-13) alternative macrophage activation and M2 phenotypic marker expression. Further investigation is required to determine whether this chemokine contributes to the pathogenesis of toxoplasmosis and to elucidate the underlying molecular mechanisms.

Burkholderia pseudomallei BopE suppresses the Rab32-dependent defense pathway to promote its intracellular replication and virulence.

Melioidosis is a serious infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Recently, Rab32-dependent immune vesicles emerge as a critical defense pathway to restrict the intracellular B. pseudomallei. However, B. pseudomallei can evade host immune vesicles and survive in the cytoplasm, although this mechanism is not well understood. In this study, we found Rab32-dependent vesicles could effectively combat B. pseudomallei infection, but not all intracellular B. pseudomallei were encapsulated in Rab32-positive vesicles. To explore how B. pseudomallei counteracted the Rab32-dependent defense pathway, transcriptomic profiling of B. pseudomallei was performed to characterize the response dynamics during infection. We found that the type III secretion system of B. pseudomallei was activated, and a variety of effector proteins were highly upregulated. Among them, BopE, BprD, and BipC were shown to interact with Rab32. Interestingly, BopE directly interacts with host Rab32, potentially suppressing Rab32 function by interfering with nucleotide exchange, which in turn restricts the recruitment of Rab32 to bacterial-containing vesicles. Knocking out of BopE can increase the proportion of Rab32-positive vesicles, suppressing the intracellular replication and virulence of B. pseudomallei. Collectively, our findings have demonstrated that BopE may be an important effector for B. pseudomallei to evade from the Rab32-dependent killing vesicles into the cytosol for survival and replication. Therefore, a deeper understanding of the interaction between BopE and the host Rab32-dependent restriction pathway may provide an effective therapeutic strategy for the elimination of intracellular B. pseudomallei.IMPORTANCEB. pseudomallei is facultative intracellular bacterium that has evolved numerous strategies to evade host immune vesicles and survive in the cytoplasm. Rab32-dependent vesicles are one of these immune vesicles, but the mechanism by which B. pseudomallei escape Rab32-dependent vesicles remains elusive. Here, we find B. pseudomallei infection leading the activation of the type III secretion system (T3SS-3) and increasing the expression of various effectors. Specifically, we identify that BopE, an effector secreted by T3SS-3, triggers vesicle escape to promote B. pseudomallei pathogenicity and survival. Mechanistically, BopE suppresses the activation of Rab32 by interfering with nucleotide exchange, ultimately triggering vesicle escape and intracellular survival. We also find knocking out the bopE gene can increase the proportion of Rab32-positive vesicles that trap B. pseudomallei, dampening the survival of B. pseudomallei both in vitro and in vivo. Taken together, our findings provide insights into the molecular mechanisms of pathogen effector-induced vesicle escape, indicating a potential melioidosis treatment via blocking B. pseudomallei BopE-host Rab32 interaction.

Edwardsiella piscicida promotes mitophagy to escape autophagy-mediated antibacterial defense in teleost monocytes/macrophages

Edwardsiella piscicida is a Gram-negative intracellular pathogen. Currently, the immune evasion strategies of E. piscicida are not yet fully understood. In this study, we revealed that E. piscicida exploited mitophagy to enhance its intracellular survival in grass carp (Ctenopharyngodon idella) monocytes/macrophages. The results showed that in E. piscicida-infected cells, there was a significant loss of mitochondrial membrane potential and a marked increase in the production of mitochondrial reactive oxygen species (mtROS), indicating the damage to the mitochondria in the infected cells. Furthermore, fluorescence confocal images presented that E. piscicida infection elicited the colocalization of mitochondria with microtubule-associated protein 1 light chain 3 (LC3, a key marker molecule of autophagy) and lysosomes, suggesting the occurrence of mitophagy. This notion was supported by the findings that E. piscicida significantly stimulated LC3-II protein and damped Tom20 (a marker molecule of mitochondria) protein levels. Subsequently, a mitophagy inducer carbonyl cyanide m-chlorophenyl hydrazine (CCCP) and a mitophagy inhibitor mitochondrial division inhibitor 1 (Mdivi-1) were used to evaluate the role of mitophagy in modulating the intracellular survival of E. piscicida. The results revealed that CCCP significantly promoted intracellular survival of E. piscicida, whereas Mdivi-1 hindered the bacterial growth. Moreover, Mdivi-1 further increased the production of bacteria-induced mitochondrial ROS in an additive manner. In particular, CCCP suppressed and Mdivi-1 promoted the colocalization of LC3-II with E. piscicida, implying that E. piscicida-triggered mitophagy to facilitate its growth probably by subverting the autophagy targeting this bacterium, a previously known clearance pathway of E. piscicida. Overall, our findings suggest a new survival strategy employed by E. piscicida within fish monocytes/macrophages.

A novel method of Francisella infection of epithelial cells using HeLa cells expressing fc gamma receptor

BackgroundFrancisella tularensis, the causative agent of tularemia, is a facultative intracellular bacterium. Although the life cycle of this bacterium inside phagocytic cells (e.g., macrophages, neutrophils) has been well analyzed, the difficulty of gene silencing and editing genes in phagocytic cells makes it difficult to analyze host factors important for the infection. On the other hand, epithelial cell lines, such as HeLa, have been established as cell lines that are easy to perform gene editing. However, the infection efficiency of Francisella into these epithelial cells is extremely low.MethodsIn order to facilitate the molecular biological analysis of Francisella infection using epithelial cells, we constructed an efficient infection model of F. tularensis subsp. novicida (F. novicida) in HeLa cells expressing mouse FcγRII (HeLa-FcγRII), and the system was applied to evaluate the role of host GLS1 on Francisella infection.ResultsAs a result of colony forming unit count, HeLa-FcγRII cells uptake F. novicida in a serum-dependent manner and demonstrated an approximately 100-fold increase in intracellular bacterial infection compared to parental HeLa cells. Furthermore, taking advantage of the gene silencing capability of HeLa-FcγRII cells, we developed GLS1, a gene encoding glutaminase, knockdown cells using lentiviral sh RNA vector and assessed the impact of GLS1 on F. novicida infection. LDH assay revealed that GLS1-knockdown HeLa-FcγRII cells exhibited increased cytotoxicity during infection with F. novicida compared with control HeLa-FcγRII cells. Furthermore, the cell death was inhibited by the addition of ammonia, the metabolite produced through glutaminase activity. These results suggest that ammonia plays an important role in the proliferation of F. novicida.ConclusionsIn this report, we proposed a new cell-based infection system for Francisella infection using HeLa-FcγRII cells and demonstrated its effectiveness. This system has the potential to accelerate cell-based infection assays, such as large-scale genetic screening, and to provide new insights into Francisella infection in epithelial cells, which has been difficult to analyze in phagocytic cells.