Intracellular Survival Research Articles

Deciphering pathogenicity and virulence of the first Staphylococcus debuckii isolate from diabetic foot osteomyelitis

IntroductionThis study identifies Staphylococcus debuckii as a new coagulase-negative staphylococcal species isolated from diabetic foot osteomyelitis (DFOM) and provides an in-depth analysis of its pathogenic and virulence profile, as well as demonstrating its potential to cause infection.MethodsThe S. debuckii NSD001 strain was examined for its planktonic growth, biofilm production, and phagocytosis rates in murine macrophages compared to S. aureus NSA739. Additionally, persistence and replication within human osteoblasts were investigated, while the zebrafish embryo model was employed to assess virulence. Genomic sequencing and bioinformatic analysis were also conducted to identify genes associated with virulent potential.Results and DiscussionS. debuckii NSD001 exhibited robust planktonic growth and significant biofilm production, highlighting its capacity to initiate and maintain an infection, and demonstrated similar rates of phagocytosis as S. aureus NSA739 in murine macrophages, suggesting a mechanism for evading initial host defenses. The strain persisted and replicated within human osteoblasts, indicative of a strategy for intracellular survival and facilitation of chronic osteomyelitis. The zebrafish embryo model revealed a slower, yet fatal, virulence profile for S. debuckii NSD001 compared to the rapid lethality induced by S. aureus NSA739. Genomic sequencing and bioinformatic analysis uncovered various genes corroborating its virulence. S. debuckii NSD001 poses a significant concern in DFOM due to its ability to form biofilms and survive within host cells, presenting challenges for current treatment strategies. This underscores the need for updated clinical protocols and increased awareness among healthcare professionals to effectively manage infections caused by this emerging pathogen.

Intraperitoneal Treatment of Cambinol, a Synthetic SIRT1 and SIRT2 Inhibitory Compound, Exacerbates Brucella abortus 544 Burden in the Spleens of Institute of Cancer Research Mice

Our preliminary data using bone marrow-derived macrophages (BMDMs) collected from ICR mice treated with anti-sirtuin (anti-SIRT) 1 antibody showed that Brucella uptake was significantly attenuated. We then further investigated the effect of an inhibitor of SIRT1/2, cambinol, in the progression of Brucella. The in vitro results using RAW264.7 cells revealed that cambinol treatment had no effect on adhesion, uptake, intracellular survival and nitric oxide (NO) production during B. abortus infection, nor did it directly affect bacterial growth for up to 72 h. Finally, intraperitoneal treatment of 8-week-old female ICR mice infected with Brucella showed no differences in the total average weights of spleens and livers; however, the treated mice displayed higher Brucella colony-forming units (CFUs) from the spleens. Furthermore, the interleukin (IL)-10 serum level was observed to be lower in treated mice at 7 d post-infection, and none of the cytokines tested showed a change at 14 d post-infection. The overall findings showed that cambinol treatment had no effect on the proliferation of Brucella in RAW264.7 macrophages but exacerbated the splenic proliferation of the bacteria in mice and displayed reduced anti-inflammatory cytokine IL-10 at the first week of infection, suggesting that cambinol as an inhibitory of SIRT1/2 could be beneficial in the context of Brucella dissemination in animal hosts and that exploration of activating SIRTs could be an alternative treatment against Brucella infection.

Whole genome sequencing reveals circulation of potentially virulent Listeria innocua strains with novel genomic features in cattle farm environments in Dhaka, Bangladesh.

Through the last decade, Listeria spp. has been detected in food and environmental samples in Bangladesh. However, the genomic information of this bacterium that prevails in the country remains scarce. This study analyzed the complete genome sequences of two Listeria spp. isolates obtained from cow dung and their drinking water collected from a cattle farm in Dhaka, Bangladesh. Both the isolates were identified as Listeria innocua, which shared almost identical genomic features. The genome sequences demonstrated the presence of 13 virulence genes associated with invasion (iap/cwhA, gtcA, and lpeA), surface protein anchoring (lspA), adherence (fbpA, and lap), intracellular survival (lplA1, and prsA2), peptidoglycan modification (oatA, and pdgA), and heat stress (clpC, clpE, and clpP). Additionally, the gene fosX, conferring resistance to fosfomycin, and two copper resistance-associated genes, copC and csoR, were identified in both. The genome sequences also revealed two plasmid replicons, rep25 and rep32, along with three insertion sequences [ISLmo3 (CP022021), ISLmo7 (CP006611), ISS1N (M37395)]. Notably, a composite transposon [CN_8789_ISS1N (M37395)], was detected in both L. innocua isolates, representing the first documented occurrence of this particular composite transposon in any reported Listeria species. Furthermore, the genomes contained four prophage regions [Listeria phage LP-030-2 (NC_021539), Listeria phage vB_LmoS_188 (NC_028871), Listeria phage A118 (NC_003216) and Escherichia phage RCS47 (NC_042128)]. Two CRISPR arrays were also identified, one belonging to the family type II-A. Multilocus Sequence Typing (MLST) analysis classified the L. innocua isolates of the same sequence type, ST-637. Single nucleotide polymorphism (SNP) analysis uncovered the presence of 231-340 SNPs between the L. innocua isolates and their closely related global lineage. In contrast, only 42 SNPs were identified between the two isolates, suggesting a potential transmission of L. innocua between cow dung and cattle farm water. The presence of L. innocua isolates harboring virulence genes associated with ruminant infection in the cattle farm environment of Bangladesh raises significant concerns about the potential presence of other human and animal pathogens. This poses a serious threat to the cattle farming industry. Additionally, the genomic analysis of the L. innocua isolates enhances our understanding of the evolutionary dynamics of Listeria species.

Comparative genomics and virulence potential of Campylobacter coli strains isolated from different sources over 25 years in Brazil

BackgroundCampylobacter spp. have been reported as a common cause of gastroenteritis in humans in many countries. However, in Brazil there is insufficient data to estimate the impact of Campylobacter in public health. In light of the importance of this foodborne pathogen, the aim of this study was to perform comparative analyses on 80 Brazilian Campylobacter coli genomes isolated from human feces, animals, the environment, and food. Methods include Average Nucleotide Identity (ANI), Gegenees, genomic plasticity, presence of pathogenicity, resistance, and metabolic islands. In addition, virulence analysis in Galleria mellonella were also performed for 18 selected C. coli strains.ResultsThe ANI values confirmed that all strains belonged to the C. coli species. Phylogenetic analyses demonstrated the evolutionary relationships among the studied strains, highlighting the genetic diversity among them. The differences in shared and deleted regions of the studied genomes were demonstrated, with 16 genomic islands identified, including 4 metabolic islands, 4 resistance islands, and 8 pathogenicity islands. We detected genes associated with chemotaxis, exotoxin production, antimicrobial resistance, stress response, defense mechanisms, and intracellular survival among these islands, highlighting the pathogenic potential of these strains. Two strains isolated from human and one from animal showed high virulence, killing 100% of Galleria mellonella larvae. Two strains isolated from the environment and two isolated from food killed 70–90% of the larvae and were classified as virulent. Three strains isolated from animal, two from human, two from the environment and one from food killed 30% to 60% of the larvae and were considered of intermediate virulence. Campylobacter jejuni ATCC 33291, one strain isolated from human and one from food killed 10 to 20% of the larvae and were considered of low virulence. One strain isolated from food did not kill any larvae and was considered avirulent.ConclusionsThe results obtained highlighted the genetic diversity, pathogenic and virulence potential of many of the C. coli strains studied, contributing for a more complete characterization of this important pathogen recognized as a cause of human gastroenteritis.

DNA damage repair factor Rad18 controls virulence partially via transcriptional suppression of genes HWP1 and ECE1 in Candida albicans

ABSTRACT DNA damage repair is a crucial cellular mechanism for rectifying DNA lesions arising during growth and development. Among the various repair pathways, postreplication repair (PRR) plays a pivotal role in resolving single-stranded gaps induced by DNA damage. However, the contribution of PRR to virulence remains elusive in the fungal pathogen Candida albicans (C. albicans). In this study, we investigated the role of Rad18, a critical component of PRR, in DNA damage response and virulence in C. albicans. We observed that deletion of RAD18 in C. albicans resulted in heightened sensitivity to DNA damage stress. Through deletion of specific internal domains coupled with spot assay analysis, we show that the internal RING and SAP domains play essential roles in DNA damage response, whereas the ZNF domain was less important. Surprisingly, the lack of Rad18 in C. albicans resulted in heightened intracellular survival within macrophages and elevated virulence in the Galleria mellonella model. RNAseq analysis revealed that loss of Rad18 upregulated the transcription of genes encoding transporters and oxidoreductases, as well as virulence genes, including HWP1 and ECE1. Suppression of the transcription of these virulence genes in the RAD18 deletion strain by a dCas9-mediated CRISPRi system reversed this increased virulence. Taken together, these data demonstrate that Rad18 plays a significant role in virulence partially through transcriptional suppression of virulence genes HWP1 and ECE1 in C. albicans. Our findings provide valuable insights into the intricate relationship between DNA damage response and virulence in C. albicans.

The T4bSS of Legionella features a two-step secretion pathway with an inner membrane intermediate for secretion of transmembrane effectors.

To promote intracellular survival and infection, Legionella spp. translocate hundreds of effector proteins into eukaryotic host cells using a type IV b protein secretion system (T4bSS). T4bSS are well known to translocate soluble as well as transmembrane domain-containing effector proteins (TMD-effectors) but the mechanisms of secretion are still poorly understood. Herein we investigated the secretion of hydrophobic TMD-effectors, of which about 80 were previously reported to be encoded by L. pneumophila. A proteomic analysis of fractionated membranes revealed that TMD-effectors are targeted to and inserted into the bacterial inner membranes of L. pneumophila independent of the presence of a functional T4bSS. While the T4bSS chaperones IcmS and IcmW were critical for secretion of all tested TMD-effectors, they did not influence inner membrane targeting of these proteins. As for soluble effector proteins, translocation of all investigated TMD-effectors depended on a C-terminal secretion signal. A deeper analysis of the TMD-effector SidF showed that this signal needed to be presented towards the cytoplasmic side of the inner membrane and that a small periplasmic loop was required for efficient translocation. We propose that strongly hydrophobic TMD-effectors are secreted in a two-step secretion process: Initially, an inner membrane intermediate is formed, that is extracted towards the cytoplasmic side, possibly by the help of the type IV coupling protein complex and subsequently secreted into eukaryotic host cells by the T4bSS core complex. Overall, our study highlights the amazing versatility of T4bSS to secrete soluble and TMD-effectors from different subcellular locations of the bacterial cell.

Inhibiting lipid droplet biogenesis enhances host protection against hypervirulent Klebsiella pneumoniae infections

Hypervirulent Klebsiella pneumoniae (hvKp), an emerging Kp subtype, has become a serious global pathogen. However, the information regarding host interactions and innate immune responses during hvKp infection is limited. Here, we found that hvKp clinical strains increased triacylglycerol synthesis, resulting in lipid droplets (LDs) formation via the mammalian target of rapamycin signaling pathway in RAW264.7 cells. Treatment with rapamycin, an inhibitor of this pathway, affected LDs formation and antimicrobial responses against clinical hvKp infections. In accordance with the role of LDs in modulating inflammation, the pharmacological inhibition of lipogenesis reduced proinflammatory cytokine expression during hvKp infections. In addition, inhibition of LDs formation using pharmacological inhibitors and knockdown of lipogenesis regulators decreased the intracellular survival of hvKp in macrophages. Moreover, inhibiting LDs biogenesis reduced mortality, weight loss, and bacterial loads in hvKp-infected mice. Collectively, these data suggest that LDs biogenesis is crucial in linking host immune responses to clinical hvKp infections.

Mycoplasma bovis activates apoptotic caspases to suppress xenophagy for its intracellular survival

Mammalian caspases are categorized into apoptotic and inflammatory types. Apoptotic caspases mediate apoptosis activation, while inflammatory caspases participate in inflammasome activation. Previous studies have shown that apoptotic caspases regulate autophagy in both cancer and pharmacological treatment models. However, the relationship between apoptotic caspases and xenophagy during pathogen infection remains elusive. In the current study, we used Mycoplasma bovis (M. bovis) as a model pathogen investigating the relationship between apoptotic caspases and xenophagy during infection. We found that M. bovis activated apoptotic caspases by triggering mitochondrial damage in macrophages, and the intracellular survival of M. bovis was enhanced by the activation of apoptotic caspases and restricted by the inhibition of apoptotic caspases. Moreover, confocal microscopy and Western blot analysis revealed that the activation of apoptotic caspases impedes host xenophagy by cleaving autophagy-related protein Beclin 1. Our findings indicate that M. bovis utilizes host apoptotic caspases to suppress xenophagy, thereby enhancing its intracellular survival. This research contributes to understanding the interplay between apoptotic caspases and xenophagy during pathogen infection, offering novel insights into the intracellular survival mechanisms of mycoplasma in macrophages.

Race for the surface between THP-1 macrophages and Staphylococcus aureus on various titanium implants with well-defined topography and wettability.

Gristina et al. (1987) suggested the fate of a biomaterial is decided in a “race for the surface” between pathogens and the host. To gain deeper insight into the mechanisms behind this concept, we investigated the “race for the surface” across three co-culture scenarios with THP-1 macrophages and Staphylococcus aureus (1:1 ratio), varying the order of addition: (i) simultaneous, (ii) macrophages first, and (iii) S. aureus first, on six Ti6Al4V-ELI surfaces modified with specific topographies and wettability. The outcome of the race for the surface was not influenced by these biomaterials but by the chronological introduction of macrophages and S. aureus. When macrophages and S. aureus arrived simultaneously, macrophages won the race, leading to the lowest number of viable S. aureus through rapid phagocytosis and killing. When macrophages arrived and established first, macrophages still prevailed but under greater challenge resulting from the lower bacterial killing efficiency of adherent macrophages and numerous viable intracellular bacteria, supporting the concept of the so-called immunocompromised zone around implants (upregulation of TLR-2 receptor and pro-inflammatory IL-1β). When S. aureus arrived first establishing a biofilm, bacteria won the race, leading to macrophage dysfunction and cell death (upregulation of FcγR and TLR-2 receptors, NF-κB signaling, NOX2 mediated reactive oxygen species), contributing to a persistent biofilm phenotype (upregulation of clfA, icaA, sarA, downregulation of agrA, hld, lukAB) and intracellular survival of S. aureus (lipA upregulation). The clinical implications are bacterial colonization of the implant and persistence of intracellular bacteria in periprosthetic tissues, which can lead to infection chronicity. Statement of SignificanceGristina et al. (1987) suggested the fate of a biomaterial is decided in a “race for the surface” between bacterial pathogens and host cells. There is a lack of in vitro co-culture models and knowledge on macrophage-S. aureus interactions on biomaterial surfaces, and none has evaluated the expression of virulence factors in S. aureus biofilms.We have successfully developed co-culture models and molecular panels and elucidated important cellular and molecular interactions between macrophages and S. aureus on a broad range of titanium biomaterials with well-defined surface topography and wettability. Our findings highlight the critical role of biofilm formation and the chronological order of bacteria or macrophage arrival in determining the fate of the surface.

Transport of miR-766–3p to A549 cells by plasma-derived exosomes and its effect on intracellular survival of Mycobacterium tuberculosis by regulating NRAMP1 expression in A549 cells

Exosomal microRNAs (miRNAs) in circulation were recognized as potential biomarkers for the diagnosis of multiple diseases. However, its potential as a diagnostic hallmark for tuberculosis (TB) has yet to be explored. Here, we comprehensively analyze miRNA profiles in exosomes derived from the plasma of active TB patients and healthy persons to evaluate its efficacy in TB diagnosis. Small-RNA transcriptomic profiling analysis identified a total of 14 differentially expressed miRNAs (DEmiRNAs), among which the diagnostic potential of exosomal miR-766–3p, miR-376c-3p, miR-1283, and miR-125a-5p was evident from their respective areas under the ROC curve, which were 0.8963, 0.8313, 0.8097, and 0.8050, respectively. The bioinformatics analysis and Luciferase reporter assays confirmed that the 3′-untranslated region of natural resistance-associated macrophage protein 1 (NRAMP1) mRNA was targeted by miR-766–3p. The exosomes could be internalized by the A549 cells in co-culturing experiments. Furthermore, both increased miR-766–3p and decreased NRAMP1 expression were observed in Mtb-infected A549 cells. MiR-766–3p overexpression reduced the NRAMP1 levels, but increased intracellular Mtb, suggesting that miR-766–3p may facilitate Mtb survival by targeting NRAMP1. Moreover, miR-766–3p-transfected cells exhibited increased apoptosis and reduced proliferation following Mtb infection. Taken together, circulating exosomal miR-766–3p, miR-1283, miR-125a-5p, and miR-376c-3p may serve as candidate hallmarks for TB diagnosis where the presence of miR-766–3p seems associated with the vulnerability to Mtb infection in humans and could be a new molecular target for therapeutic intervention of TB.

The Role of Changes in the Redox Status in the Pathogenesis of Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia is a hemoblastosis of CD5+ B lymphocytes with lymphocytosis, damage to the lymphatic organs, occurring in the older age group, the etiology and pathogenesis of which are not fully understood. Oxidative stress is an important factor in the regulation of stem cells and the activation of intracellular survival signaling pathways in chronic lymphocytic leukemia cells. The aim of the study was to analyze the current data on the role of redox status changes in the pathogenesis of chronic lymphocytic leukemia. A review of published relevant studies 2018-2023, scientific articles in scientific electronic bibliographic databases PubMed and Social Sciences Citation Index, devoted to the pathogenesis of chronic lymphocytic leukemia and the role of free-radical oxidation processes in it was carried out. In chronic lymphocytic leukemia, oxidative stress with a systemic excess of reactive oxygen species, an imbalance in the effectiveness of antioxidant defense is caused mainly by activation of oxidative phosphorylation in mitochondria, low levels of NADPH-oxidase type 2, increased expression of heme oxygenase-1, glutathione peroxidase and glutathione recycling enzymes, superoxide dismutase-2, thioredoxins and decreased expression of catalase. One of the mechanisms of resistance to drug therapy and oxidative stress of chronic lymphocytic leukemia cells is the intracellular signaling pathway dependent on erythroid nuclear factor-2, due to the activation of expression in cells of superoxide dismutase-2, catalase, glutathione peroxidase, peroxiredoxin-3 and-5, heme oxygenase-1, thioredoxin-1 and -2, reduced glutathione, natural killer cell activity, which is associated with lifespan, chemotaxis, proliferation, and survival. FOXO family proteins are believed to suppress carcinogenesis. FOXO3a increases the expression of superoxide dismutase-2, catalase, glutathione peroxidase, peroxiredoxin-3 and -5, and the activity of natural killer cells, which promotes the survival of tumor cells. The development of new targeted pharmacological agents that are capable of accumulating reactive oxygen species and reducing antioxidant protection due to the degradation of erythroid nuclear factor-2 and activation of NADPH-quinone oxidoreductase-1 is underway, which modernizes the therapy of chronic lymphocytic leukemia.

Troglitazone reduces intracellular Mycobacterium tuberculosis survival via macrophage autophagy through LKB1-AMPKα signaling.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb), results in significant morbidity and mortality worldwide. Host-directed therapy (HDT), including conventional drugs, is a promising anti-TB strategy that shows synergistic antibacterial effects when combined with anti-TB drugs. Here, the mycobactericidal effect of three anti-diabetic drugs was examined. Of these, only Troglitazone (Trog) enhanced the antimycobacterial effect in vitro and in vivo. This was due to Trog-mediated autophagy activation. Moreover, a knock-down experiment revealed that Trog activated autophagy and exhibited antimycobacterial activity through the LKB1-AMPK signaling pathway. Molecular docking and co-immunoprecipitation experiments demonstrated that Trog promoted LKB1 phosphorylation and activation by targeting STRADA. Finally, we found that Trog inhibited the intracellular survival of clinical isoniazid (INH)-resistant Mtb, and the combination of Trog and INH showed additive antibacterial effects against Mtb H37Rv. Taken together, anti-diabetic Trog may be repurposed as an HDT candidate and combined with first-line anti-TB drugs.

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.

Hypermigration of macrophages through the concerted action of GRA effectors on NF-κB/p38 signaling and host chromatin accessibility potentiates Toxoplasma dissemination.

Intracellular pathogens can hijack the cellular functions of infected host cells to their advantage, for example, for intracellular survival and dissemination. However, how microbes orchestrate the hijacking of complex cellular processes, such as host cell migration, remains poorly understood. As such, the common parasite Toxoplasma gondii actively invades the immune cells of humans and other vertebrates and modifies their migratory properties. Here, we show that the concerted action of a number of secreted effector proteins from the parasite, principally GRA15 and GRA24, acts on host cell signaling pathways to activate chemotaxis. Furthermore, the protein effector GRA28 selectively acted on chromatin accessibility in the host cell nucleus to selectively boost host gene expression. The joint activities of GRA effectors culminated in pro-migratory signaling within the infected phagocyte. We provide a molecular framework delineating how T. gondii can orchestrate a complex biological phenotype, such as the migratory activation of phagocytes to boost dissemination.

Functional and Proteomic Dissection of the Contributions of CodY, SigB and the Hibernation Promoting Factor HPF to Interactions of Staphylococcus aureus USA300 with Human Lung Epithelial Cells.

Staphylococcus aureus is a leading cause of severe pneumonia. Our recent proteomic investigations into S. aureus invasion of human lung epithelial cells revealed three key adaptive responses: activation of the SigB and CodY regulons and upregulation of the hibernation-promoting factor SaHPF. Therefore, our present study aimed at a functional and proteomic dissection of the contributions of CodY, SigB and SaHPF to host invasion using transposon mutants of the methicillin-resistant S. aureus USA300. Interestingly, disruption of codY resulted in a "small colony variant" phenotype and redirected the bacteria from (phago)lysosomes into the host cell cytoplasm. Furthermore, we show that CodY, SigB and SaHPF contribute differentially to host cell adhesion, invasion, intracellular survival and cytotoxicity. CodY- or SigB-deficient bacteria experienced faster intracellular clearance than the parental strain, underscoring the importance of these regulators for intracellular persistence. We also show an unprecedented role of SaHPF in host cell adhesion and invasion. Proteomic analysis of the different mutants focuses attention on the CodY-perceived metabolic state of the bacteria and the SigB-perceived environmental cues in bacterial decision-making prior and during infection. Additionally, it underscores the impact of the nutritional status and bacterial stress on the initiation and progression of staphylococcal lung infections.

Suppressive effects of Toll-Like Receptor 2, Toll-Like Receptor 4, and Toll-Like Receptor 7 on protective responses to Mycobacterium bovis BCG from epithelial cells

Mycobacteria have several mechanisms for evasion of protective responses mounted by the host. In this study, we unravel yet another mechanism that is mediated by Toll-Like Receptors TLR2, TLR4, and TLR7 in epithelial cells. We show that mycobacterial infection of epithelial cells increases the expression of TLR2, TLR4, and TLR7. Stimulation of either TLR along with mycobacterial infection results in an inhibition of oxidative burst resulting in increased survival of mycobacteria inside epithelial cells. TLR stimulation along with mycobacterial infection also inhibits activation of epithelial cells for T cell responses by differentially regulating the activation of ERK-MAPK and p38-MAPK along with inhibition of co-stimulatory molecule CD86 expression. Furthermore, stimulation of either TLR inhibits the induction of apoptosis and autophagy. Knockdown of either TLR by specific siRNAs reverses the inhibition by ROS and apoptosis by mycobacteria and results in reduced intracellular survival of mycobacteria in a MyD88-dependent manner. These results point towards a negative role for TLR2, TLR4, and TLR7 in regulating protective responses to M. bovis BCG infection in epithelial cells.

Mycobacterium manipulate glutaminase 1 mediated glutaminolysis to regulate macrophage autophagy for bacteria intracellular survival

Autophagy plays a vital role in eliminating intracellular mycobacterium. It is regulated by multiple metabolic processes including glutaminolysis. Glutaminase 1 (GLS1) is the rate-limiting enzyme of glutaminolysis and has been reported to control intracellular Gln content. However, its function on regulating autophagy in mycobacterium infected macrophage is still obscure. Hence, the current study hired mycobacterium virulent strain H37Rv or attenuated strain BCG to infect macrophage and detected the changes in cell glutaminolysis. The function of GLS1 on regulating autophagy in mycobacterium infected macrophages was further investigated. The results showed that BCG infection promoted macrophage autophagy, enhanced glutaminolysis, reduced intracellular Gln content, accompanied with the up-regulation of GLS1. Conversely, H37Rv infection resulted in completely opposite effects. Meanwhile, knockdown of GLS1 increased Gln content and attenuated autophagy in BCG infected macrophages. In addition, the deprivation of Gln not only promoted the autophagy of H37Rv infected macrophages, but also abolished the effect of knockdown GLS1 on regulating BCG infection-induced mTOR activation or autophagy. To sum up, our study suggested that different virulent strains of mycobacterium infection have totally opposite effects on glutaminolysis and the expression of GLS1. Specifically, mycobacterium virulent strain reduced GLS1 expression and decreased Gln content but mycobacterium attenuated strain promoted GLS1 expression and enhanced Gln content. Furthermore, GLS1 inhibits the activation of the mTOR signaling pathway and promotes autophagy by decreasing Gln content.

Toxoplasma acyl-CoA synthetase TgACS3 is crucial to channel host fatty acids in lipid droplets and for parasite propagation

Apicomplexa comprise important pathogenic parasitic protists that heavily depend on lipid acquisition to survive within their human host cells. Lipid synthesis relies on the incorporation of an essential combination of fatty acids (FAs) either generated by a metabolically adaptable de novo synthesis in the parasite or by scavenging from the host cell. The incorporation of FAs into membrane lipids depends on their obligate metabolic activation by specific enzyme groups, acyl-CoA synthetases (ACSs). Each ACS has its own specificity, so it can fulfill specific metabolic functions. Whilst such functionalities have been well studied in other eukaryotic models, their roles and importance in Apicomplexa are currently very limited, especially for Toxoplasma gondii. Here, we report the identification of seven putative ACSs encoded by the genome of T. gondii (TgACS), which localize to different sub-cellular compartments of the parasite, suggesting exclusive functions. We show that the perinuclear/cytoplasmic TgACS3 regulates the replication and growth of Toxoplasma tachyzoites. Conditional disruption of TgACS3 shows that the enzyme is required for parasite propagation and survival, especially under high host nutrient content. Lipidomic analysis of parasites lacking TgACS3 reveals its role in the activation of host-derived FAs that are used for i) parasite membrane phospholipid and ii) storage triacylglycerol (TAG) syntheses, allowing proper membrane biogenesis of parasite progenies. Altogether, our results reveal the role of TgACS3 as the bulk FA activator for membrane biogenesis allowing intracellular division and survival in T. gondii tachyzoites, further pointing to the importance of ACS and FA metabolism for the parasite.

Mycobacterium marinum MMAR_0267-regulated copper utilization facilitates bacterial escape from phagolysosome

The host limits Mycobacterium tuberculosis (Mtb) by enriching copper in high concentrations. This research investigates how Mtb escapes copper stress. The membrane protein encoded by Mtb Rv0102, when its homolog in M. smegmatis (MSMEG_4702) was knocked out, resulted in a fourfold decrease in intracellular copper levels and enhanced tolerance to elevated extracellular copper concentrations. Similarly, knockout mutants of its homolog in M. marinum (MMAR_0267) showed increased virulence in zebrafish and higher bacterial load within macrophages. In THP-1 cells infected with MMAR_0267 deletion mutants, the intracellular survival of these mutants increased, along with reduced THP-1 cell apoptosis. Deficiency in copper down-regulated the transcriptional level of the virulence factor CFP-10 in M. marinum, suppressed cytosolic signaling via the macrophage STING pathway, leading to decreased production of IFN-β and reduced cell apoptosis. In conclusion, these findings highlight the significant impact of copper on the survival and reproduction of mycobacteria, underscoring the importance of studying mycobacterial adaptation mechanisms in copper-rich environments.