Virulence

Tetraspanins (TSPANs) are a family of proteins highly conserved in all eukaryotes. Although protein-protein interactions of TSPANs have been well established in eukaryotes including parasitic protists, the role they play in parasitism and pathogenesis remains largely unknown. In this study, we characterized three representative members of TSPANs, TSPAN4, TSPAN12, and TSPAN13 from the human intestinal protozoan Entamoeba histolytica. Co-immunoprecipitation assays demonstrated that TSPAN4, TSPAN12 and TSPAN13 are reciprocally pulled down together with several other TSPAN-interacting proteins including TSPAN binding protein of 55kDa (TBP55) and interaptin. Blue native PAGE analysis showed that these TSPANs form several complexes of 120-250 kDa. Repression of tspan12 and tspan13 gene expression led to decreased secretion of cysteine proteases. Meanwhile, strains overexpressing HA-tagged TSPAN12 and TSPAN13 demonstrated reduced adhesion to collagen. Altogether, this study reveals that the TSPANs, especially TSPAN12 and TSPAN13, are engaged with complex protein-protein interactions and are involved in the pathogenicity-related biological functions such as protease secretion and adhesion, offering insights into the potential regulatory mechanisms of tetraspanins in protozoan parasites.

Microsporidia are a group of intracellular pathogens that actively manipulate host cell biological processes to facilitate their intracellular niche. Apoptosis is an important defense mechanism by which host cell control intracellular pathogens. Microsporidia modulating host cell apoptosis has been reported previously, however the molecular mechanism is not yet clear. In this report, we describe that the microsporidia Nosema bombycis inhibits apoptosis of Bombyx mori cells through a secreted protein NbSPN14, which is a serine protease inhibitor (Serpin). An immunofluorescent assay demonstrated that upon infection with N. bombycis, NbSPN14 was initially found in the B. mori cell cytoplasm and then became enriched in the host cell nucleus. Overexpression and RNA-interference (RNAi) of NbSPN14 in B. mori embryo cells confirmed that NbSPN14 inhibited host cell apoptosis. Immunofluorescent and Co-IP assays verified the co-localization and interaction of NbSPN14 with the BmICE, the caspase 3 homolog in B. mori. Knocking out of BmICE or mutating the BmICE-interacting P1 site of NbSPN14, eliminated the localization of NbSPN14 into the host nucleus and prevented the apoptosis-inhibiting effect of NbSPN14, which also proved that the interaction between BmICE and NbSPN14 occurred in host cytoplasm and the NbSPN14 translocation into host cell nucleus is dependent on BmICE. These data elucidate that N. bombycis secretory protein NbSPN14 inhibits host cell apoptosis by directly inhibiting the caspase protease BmICE, which provides an important insight for understanding pathogen-host interactions and a potential therapeutic target for N. bombycis proliferation. Author SummaryMicrosporidia constitute a class of eukaryotic pathogens that exclusively reside within host cells. The species Nosema bombycis is the first microsporidian identified as the pathogen of silkworm Pebrine disease. In our research, we discovered how N. bombycis cleverly evades the hosts defenses. It has developed a strategy to survive inside host cells by manipulating host cell apoptosis, disarming the host cells self-destruct mechanism. In this study, we discovered that the N. bombycis secretes a serine protease inhibitor named NbSPN14, which infiltrates the cytoplasm of the host cell. The NbSPN14 interacts with the executioner Caspase protease BmICE within the silkworms apoptotic pathway, effectively neutralizing its apoptoic activity and thus curbing the apoptosis of the host cells.

Salmonella is a facultative intracellular pathogen that has co-evolved with its host and has also developed various strategies to evade the host immune responses. Salmonella recruits an array of virulence factors to escape from host defense mechanisms. Previously chitinase A (chiA) was found to be upregulated in intracellular Salmonella. Although studies show that chitinases and chitin binding proteins (CBP) of many human pathogens have a profound role in various aspects of pathogenesis, like adhesion, virulence and immune evasion, the role of chitinase in strict intravacuolar pathogen Salmonella has not yet been elucidated. In this study, we deciphered the role of chitinase of Salmonella in the pathogenesis of the serovars, Typhimurium and Typhi. Our data propose that ChiA mediated modification of the glycosylation on the epithelial cell surface facilitates the invasion of the pathogen into the epithelial cells. Further we found that ChiA aids in reactive nitrogen species (RNS) and reactive oxygen species (ROS) production in phagocytes, leading to MHCII downregulation followed by suppression of antigen presentation and antibacterial responses. In continuation of the study in animal model C. elegans, Salmonella Typhi ChiA was found to facilitate attachment to the intestinal epithelium, gut colonization and persistence by downregulating antimicrobial peptides.

Cutibacterium acnes is a pathogenic bacterium that cause inflammatory diseases of the skin and intervertebral discs. The immune activation induced by C. acnes requires multiple cellular responses in the host. Silkworm, an invertebrate, generates melanin by phenoloxidase upon recognizing bacterial or fungal components. Therefore, the melanization reaction can be used as an indicator of innate immune activation. A silkworm infection model was developed for evaluating the virulence of C. acnes, but a system for evaluating the induction of innate immunity by C. acnes using melanization as an indicator has not yet been established. Here we demonstrated that C. acnes rapidly causes melanization of the silkworm hemolymph within 3 h. On the other hand, Staphylococcus aureus, a gram-positive bacterium identical to C. acnes, does not cause immediate melanization. Even injection of heat-killed C. acnes cells caused melanization of the silkworm hemolymph. DNase, RNase, and protease treatment of the heat-treated C. acnes cells did not decrease the silkworm hemolymph melanization. Treatment with peptidoglycan-degrading enzymes, such as lysostaphin and lysozyme, however, decreased the induction of melanization by the heat-treated C. acnes cells. These findings suggest that silkworm hemolymph melanization may be a useful indicator to evaluate innate immune activation by C. acnes and that C. acnes peptidoglycans are involved in the induction of innate immunity in silkworms.

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 the 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). Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. A report showed that Salmonella containing vacuole (SCV) harbors recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis is unclear. Upon knockdown of STX3, we have observed a reduction in bacterial proliferation and is restored upon the overexpression of STX3. Post infected live-cell imaging of cells showed STX3 localises to the SCV membranes and thus might help in fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found this interaction abrogated when we infected with SPI-2 encoded T3SS apparatus mutant (STM {Delta}ssaV) but not with SPI-1 encoded T3SS (STM{Delta} invC). These observations were also consistent in mice model of Salmonella infection. Together, these results shed a light on the effector molecules secreted through SPI-2 encoded by T3SS possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and maintenance the principle single bacterium per vacuole. SynopsisSalmonella Typhimurium infection in murine macrophage leads to upregulation of host Syntaxin 3 both at transcript and protein levels at late stage of infection. Syntaxin 3 cross-talk with Salmonella containing vacuoles (SCVs) is essential for establishment of replicative niche in host macrophages. The cross-talk between STX3 and SCVs is Salmonella pathogenicity island 2 (SPI-2) dependent and is consistent in mice model of Salmonella Typhimurium infection.

Microsporidia are difficult to completely eliminate. Their persistence may disrupt host cell functions. Here in this study, we aimed to elucidate the impairing effects and consequences of microsporidia infection upon dendritic cells (DCs). We used the zoonotic microsporidia species, Enterocytozoon hellem, in our studies. In vivo experiments showed that E. hellem-infected mice were more susceptible to further pathogenic challenges. DCs were identified as the most affected group of cells. In vitro assays revealed that E. hellem infection impaired the immune functions of DCs as reflected by down-regulation of cytokine expression, lower extent of maturation and antigen presentation. E. hellem infection decreased the ability of DCs to prime and stimulate T cells, thereby hampering host immune cell functions. We further demonstrate that E. hellem Ser/Thr protein phosphatase PP1 directly interacts with host p38(MAPK14) to manipulate the p38 (MAPK14)/NFAT-5 axis of the MAPK pathway. Our study is the first to elucidate the molecular mechanisms of the impairing effects of microsporidia on host DCs immune functions. The emerging of microsporidiosis may be great threat to public health. HighlightsO_LIPersistence of Microsporidia within host impairs dendritic cell functions such as phagocytosis, maturation, antigen presentation and T cell priming, thereby disrupting both innate and adaptive immunities and making the host more vulnerable to secondary infections C_LIO_LIMicrosporidia impairs DCs function via Serine/Threonine Protein Phosphatase PP1 directly targeting DCs p38/MAPK pathway C_LIO_LILatent Microsporidia infection and persistence is a great threat to public health when assessing acute and emerging pathogen risk C_LI

Autophagy has emerged as a critical innate immune mechanism for host elimination of intracellular pathogens, however, the role of the autophagy receptor optineurin during mycobacterial infection is not fully understood. To address this lacuna, we infected bone marrow-derived macrophages (BMDMs) derived from Optn+/+ and Optn-/- mice with Mycobacterium smegmatis, and observed the infection outcome at sequential time points. While low multiplicity of infection (MOI) did not show any significant difference between BMDMs from the two groups, at high MOI Optn-/- mice-derived macrophages showed significantly lower colony forming unit counts, as well as lower cell counts at 12 h and 24 h post-infection. Quantification of cell numbers and nuclear morphologies at various time points post-infection indicated a markedly higher cell death in the optineurin-deficient macrophages. Optineurin-deficient macrophages showed significantly lower levels of the autophagosomal protein LC3-II upon infection, indicating a potential role for optineurin in regulating autophagy during mycobacterial infection. Moreover, when stimulated by bacterial LPS, optineurin deficient macrophages, showed altered levels of the inflammatory cytokine pro-IL-1{beta}. These observations taken together suggest a novel regulatory role for optineurin during mycobacterial infection, with its deficiency leading to an impairment in macrophage responses.

Sirtuins are the major players in host immuno-metabolic regulation. However, the role of sirtuins in the modulation of the immune metabolism pertaining to Salmonellosis is largely unknown. Here, our investigation focussed on the role of two important sirtuins, SIRT1 and SIRT3, shedding light on their impact on intracellular Salmonellas metabolic switch and pathogenesis establishment. Our study indicated the ability of the live Salmonella Typhimurium to differentially regulate the levels of SIRT1 and SIRT3 for maintaining the high glycolytic metabolism and low fatty acid metabolism in Salmonella. Perturbing SIRT1 or SIRT3 through knockdown or inhibition, resulted in a remarkable shift in the host metabolism to low fatty acid oxidation and high glycolysis. This switch led to decreased proliferation of Salmonella in the macrophages. Further, Salmonella-induced higher levels of SIRT1 and SIRT3 led to a skewed polarization state of the macrophages from a pro-inflammatory M1 state toward an immunosuppressive M2 making it more conducive for the intracellular life of Salmonella. Alongside, governing immunological functions by modulating p65 NF-{kappa}B acetylation, SIRT1, and SIRT3 also skew Salmonella-induced host metabolic switch by regulating the acetylation status of HIF-1 and PDHA1. Interestingly, though knock-down of SIRT1/3 attenuated Salmonella proliferation in macrophages, in in vivo mice-model of infection, inhibition or knockdown of SIRT1/3 led to more dissemination and higher organ burden which can be attributed to enhanced ROS and IL-6 production. Our study hence reports for the first time that Salmonella modulates SIRT1/3 levels to maintain its own metabolism for successful pathogenesis.

Burkholderia pseudomallei (Bpm) is the causative agent of the disease melioidosis. As a facultative intracellular pathogen, Bpm has a complex lifestyle that culminates in cell-to-cell fusion and multinucleated giant cells (MNGCs) formation. The virulence factor responsible for MNGC formation is the type 6 secretion system (T6SS), a contractile nanomachine. MNGC formation is a cell-to-cell spread strategy that allows the bacteria to avoid the extracellular immune system and our previous data highlighted cell death, apoptosis, and inflammation as pathways significantly impacted by T6SS activity. Thusly, we investigated how the T6SS influences these phenotypes within the macrophage and pulmonary models of infection. Here we report that the T6SS is responsible for exacerbating apoptotic cell death during infection in both macrophages and the lungs of infected mice. We also demonstrate that although the T6SS does not influence differential macrophage polarization, the M2 polarization observed is potentially beneficial for Bpm pathogenesis and replication. Finally, we show that the T6SS contributes to the severity of inflammatory nodule formation in the lungs, which might be potentially connected to the amount of apoptosis that is triggered by the bacteria.

Trichomonas vaginalis and Tritrichomonas foetus are extracellular flagellated parasites that inhabit humans and other mammals, respectively. In addition to motility, flagella act in a variety of biological processes in different cell types; and extra-axonemal structures (EASs) has been described as fibrillar structures that provide mechanical support and act as metabolic, homeostatic and sensory platforms in many organisms. Here, we identified the presence of EASs forming prominent flagellar swellings in T. vaginalis and T. foetus and we observed that their formation was associated with the parasites adhesion on the host cells, fibronectin, and precationized surfaces; and parasite:parasite interaction. A high number of rosettes, clusters of intramembrane particles that has been proposed as sensorial structures, and microvesicles protruding from the membrane were observed in the EASs. The protein VPS32, a member of the ESCRT-III complex crucial for diverse membrane remodeling events, the pinching off and release of microvesicles, was found in the surface as well as in microvesicles protruding from EASs. Moreover, we demonstrated that overexpression of VPS32 protein induce EAS formation and increase parasite motility in semi-solid medium. These results provide valuable data about the role of the flagellar EASs in the cell-to-cell communication and pathogenesis of these extracellular parasites.

S. aureus is a human pathogen that is extremely adaptable and is the cause of a variety of nosocomial and community-acquired infectious illnesses. During infection, S. aureus affect the host cell in many ways to enable its own multiplication, spread, and evasion of host immune defense. One of S. aureus mechanism to survive is to inhibit the SUMOylation of host proteins in order to increase its intracellular survival and persistence for longer period of time. Here, we show that the reduction in the levels of cellular SUMO-conjugated proteins is associated to the PtpA secreted virulence factor, which results in a reduction of Ubc9 protein level, the essential enzyme of the SUMOylation modification. In addition, we demonstrated that the critical residue D120A, essential for PtpA phosphatase activity, is required. This study shows for the first time that the secreted phosphatase PtpA impedes the host SUMOylation response, thus promoting S. aureus survival at long-term infection.

Brucella species are intracellular bacterial pathogens, causing the world-wide zoonotic disease, brucellosis. Brucella invade professional and non-professional phagocytic cells, followed by resisting intracellular killing and establishing a replication permissive niche. Brucella also modulate the innate and adaptive immune responses of the host for their chronic persistence. The complex intracellular cycle of Brucella majorly depends on multiple host factors but limited information is available on host and bacterial proteins that play essential role in the invasion, intracellular replication and modulation of host immune responses. By employing an siRNA screening, we identified a role for the host protein, FBXO22 in Brucella-macrophage interaction. FBXO22 is the key element in the SCF E3 ubiquitination complex where it determines the substrate specificity for ubiquitination and degradation of various host proteins. Downregulation of FBXO22 by siRNA or CRISPR-Cas9 system, resulted diminished uptake of Brucella into macrophages, which was dependent on NF-{kappa}B-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella-infected macrophages that resulted induction of phagocytic receptors and enhanced production of pro-inflammatory cytokines through NF-{kappa}B. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella, including the anti-inflammatory proteins, TcpB and OMP25 for degradation through the SCF complex. We did not observe any role for another F-box containing protein of SCF complex, {beta}-TrCP in Brucella-macrophage interaction. Our findings unravel novel functions of FBXO22 in host-pathogen interaction and its contribution to pathogenesis of infectious diseases. Author SummaryBrucellosis is a major zoonotic disease world-wide that poses a serious veterinary and public health problem in various countries, impacting their economic development. Brucellosis is caused by the species of intracellular bacterial pathogen, Brucella that replicates in professional and non-professional phagocytic cells. Brucella is considered as a stealthy pathogen as it invades/suppresses host defense responses using various virulence strategies. Brucella hijacks many cellular processes for gaining entry into the target cells, followed by establishing a replication permissive niche. However, host proteins that are involved in Brucella-macrophage interaction remains obscure. Here, we identified the host protein, FBXO22 that recruits target proteins to SCF E3 ubiquitination complex for their ubiquitination and degradation. We found that down-regulation and upregulation of FBXO22 decreased and enhanced the uptake of Brucella by macrophages, respectively. Our subsequent studies revealed that Brucella induces the expression of FBXO22 that resulted activation of NF-{kappa}B and the concomitant upregulation of phagocytic receptors that might have contributed to the enhanced uptake of Brucella. The Brucella-induced expression of FBXO22 resulted enhanced production of pro-inflammatory cytokines. We have also found that FBXO22 targets Brucella effectors, including the anti-inflammatory effector proteins for degradation through the SCF complex. Our experimental data reveals that FBXO22 plays an important role in the uptake of microbial pathogens by macrophages and pathogenesis of infectious diseases that is resulting from overt inflammatory responses.

Salmonella Typhimurium (STM) resides in a membrane-bound compartment called Salmonella containing vacuole (SCV) in several infected cell types. Within host cells, the division of bacteria and SCV are synchronous to maintain the single bacterium per vacuole. However, the mechanism regulating the synchronous fission and the machinery is not well understood. The fission of several intracellular organelles is regulated by the dynamic nature of the tubular endoplasmic reticulum (ER). In this study, we have evaluated the role of ER in controlling SCV fission. Interestingly, Salmonella-infected cells show the activation of unfolded protein response (UPR) with expanded ER tubules compared to the uninfected cells. Further, changing the expression of ER morphology regulators, such as reticulon-4a (Rtn4a) and CLIMP63, affected bacterial proliferation significantly, suggesting a potential role for tubular ER in facilitating the SCV division. Live-cell imaging analysis shows the marking of tubular ER precisely at the center of the majority of SCV division (78%) sites. We have investigated the role of SteA (a known Salmonella effector in modulating the membrane dynamics) in coordinating the SCV division. We observed that SteA resides on the SCV membranes and helps in making membrane contact sites between SCV and ER. Accordingly, the colocalization of ER with SCV enclosing SteA mutant Salmonella was significantly reduced compared to SCV-formed by wild-type Salmonella. Depletion of steA in Salmonella resulted in profound defects in SCV division, resulting in multiple bacteria residing in a single vacuole with defects in proliferation compared to the wild-type strain in epithelial cells. Also, during in vivo infection, the STM{Delta}steA mutant shows a defect in colonization in the spleen and liver and affects the initial survival rate of mice. Overall, this study suggests a coordinated role of bacterial effector SteA in promoting the ER contact sites with SCVs and thus regulating the successful division of SCV. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/592158v2_ufig1.gif" ALT="Figure 1000"> View larger version (72K): org.highwire.dtl.DTLVardef@21e83aorg.highwire.dtl.DTLVardef@157052org.highwire.dtl.DTLVardef@1815308org.highwire.dtl.DTLVardef@1724888_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

The nematode Caenorhabditis elegans has emerged as a popular model system to investigate cell biology and host-pathogen interactions. Presently, C. elegans is studied as a natural host of intracellular pathogens such as microsporidia and Orsay virus along with extracellular bacterial and fungal pathogens. The use of C. elegans as a model in host-pathogen research is limited by the number of naturally occurring pathogens to the organism. Through a sampling project to identify new pathogens of C. elegans, we identified the fungus Mucor hiemalis as a pathogen of Caenorhabditis species. We observed the fungus in the intestinal lumen of wild-caught Caenorhabditis briggsae, and co-culturing the wild-caught species with infection reporter C. elegans confirmed infection by M. hiemalis. This study characterizes the fungal infection by M. hiemalis in Caenorhabditis nematodes. Fluorescence microscopy with fungal staining revealed the life cycle of M. hiemalis within multiple Caenorhabditis species at varying growth stages. We observed the killing of nematodes by M. hiemalis via intestinal perforation and assessed its host range through a series of lifespan assays. We investigated the food preference of C. elegans and determined that nematodes show a preference towards food that contains M. hiemalis spores. Lastly, we evaluated common C. elegans transcriptional immune responses and found that M. hiemalis does not induce genes associated with the intracellular pathogen response or other responses seen with previously studied bacterial and fungal pathogens. Characterization of this fungal infection in Caenorhabditis nematodes will provide new insights into the biology of pathogenic fungi and host immune responses.

Bacterial chitinases serve to hydrolyse chitin as food source or as defence mechanism. Given that chitin is not produced by mammals, it is intriguing that Mycobacterium tuberculosis, an exclusively human pathogen harbours Rv1987, a probable chitinase and secretes it. Interestingly genes annotated as chitinases are widely distributed among Mycobacterium tuberculosis complex species, clinical isolates and other human pathogens M. abscessus and M. ulcerans. However, Mycobacterial chitinases are not characterized and hence the functions remain unknown. In the present study, we show that Rv1987 is a chitin and cellulose binding protein lacking enzymatic activity in contrary to its current annotation. Further, we show Rv1987 has moon lighting functions in M. tuberculosis pathobiology signifying roles of bacterial cellulose binding clusters in infections.

Coxiella burnetii is an obligate intracellular bacterial pathogen that causes a zoonotic disease known as Q fever. Upon internalization into host cells, Coxiella extensively remodels host vesicle trafficking and replicates within acidic, lysosome-derived, spacious vacuoles by secreting a suite of effectors through the type IVB secretion system (T4BSS), and hijacking the host cell machinery. However, how fundamental lysosomal functions, such as exocytosis, are subverted during Coxiella infection has not been well understood. In this study, we aimed to investigate the regulation and role of exocytosis in the context of Coxiella infection. Biochemical and fluorescence-based imaging approaches indicated that C. burnetii infection promotes release of extracellular vesicles (EVs). Increase in extracellular levels of endolysosomal proteins (LAMP1 and cathepsin D) and surface LAMP1 expression were identified in both phagocytic and non-phagocytic cells, during later stages of infection. Interestingly, infection-induced exocytosis was dependent on the activity of the bacterial T4BSS. Modulating the activity of TRPML1, a host calcium channel that induces exocytosis by regulating the release of calcium from lysosomes into the cytosol, using synthetic agonist/antagonist, led to a decrease in intracellular C. burnetii replication, suggesting a complex, tightly regulated role of TRPML1 during infection. Together, this study demonstrates that infection by the lysosome-adapted pathogen Coxiella burnetii activates exocytosis in a temporal, host and bacterial factors-dependent manner.

Brucella abortus is a pathogen that survives in macrophages. Several virulence factors participate in this process, including the open reading frame (ORF) BAB1_0270 codifying of a Zinc-dependent metalloproteinase. Here, its contribution in the process of intracellular adaptation was analyzed by infecting RAW264.7 macrophages with the mutant B. abortus {Delta}270 strain. Results showed that this Zinc-dependent metalloproteinase is a cytoplasmic protein that conforms an operon with a transcriptional regulator, which may constitute a type II toxin-antitoxin system. Functionally, this Zinc-dependent metalloproteinase participated neither in the adherence nor the initial intracellular traffic of B. abortus in macrophages. Nevertheless, its deletion significantly increased the co-localization of B. abortus {Delta}270 with phagolysosomal cathepsin D, reducing both its co-localization with calnexin, present in endoplasmic reticulum derived vesicles, and its intracellular replication within macrophages. Besides, B. abortus {Delta}270-infected macrophages produced significantly higher levels of TNF-, IL-6, CD80 and CD86 than B. abortus 2308, even when several genes involved in virulence (vjbR, hutC, bvrR, virB1) were up-regulated in this mutant. Finally, its deletion significantly reduced the capacity of B. abortus {Delta}270 to adapt, grow and express several virulence factors under acidic conditions. Based on these results, we discuss the role of this Zinc-dependent metalloproteinase in the regulation of the virulence of this pathogen, concluding that it contributes significantly to the intracellular adaptation of B. abortus 2308 during the infection of macrophages. Author summaryBrucella abortus is the causative agent of the brucellosis, a highly contagious diseases. A Zinc-dependent metalloproteinase contributes significantly in the intracellular survival. Here, we demonstrate that this metalloproteinase has homology with ImmA/IrrE proteases, which are involved in the bacterial resistance to hostile environment. Furthermore, it conforms a gene pair with a transcriptional regulator, being required by B. abortus to escape from phagolysosomes, to achieve the endoplasmic reticulum and replicate within macrophages. Its deletion from B. abortus stimulated the macrophages, which produced higher levels of pro-inflammatory cytokines and co-stimulatory proteins. This pathogen showed a reduced ability to adapt and grow under acidic conditions, which would negatively affect its escape from phagolysosomes and consequently, stimulating macrophages. Therefore, this work describes how this Zinc-dependent metalloproteinase significantly contributes in the intracellular adaptation of B. abortus 2308 in macrophages.

Leptospirosis, caused by pathogenic Leptospira species, has emerged as a widespread zoonotic disease worldwide. Macrophages mediate the elimination of pathogens through phagocytosis and cytokine production. Scavenger receptor A1 (SR-A1), one of the critical receptors mediating this process, plays a complicated role in innate immunity. However, the role of SR-A1 in the immune response against pathogenic Leptospira invasion is unknown. In the present study, we found that SR-A1 is an important nonopsonic phagocytic receptor on murine macrophages for Leptospira. We also found that leptospiral LPS is the ligand of SR-A1. However, intraperitoneal injection of leptospires into WT mice presented with more severe jaundice, subcutaneous hemorrhaging, and higher bacteria burdens in blood and tissues than that of SR-A1-/- mice. Exacerbated cytokine and inflammatory mediator levels were also observed in WT mice and higher recruited macrophages in the liver than those of SR-A1-/- mice. Our findings collectively reveal that although beneficial in the uptake of Leptospira by macrophage, SR-A1 might be exploited by Leptospira to promote bacterial dissemination and modulate inflammatory activation, which causes a more severe infection in the host. These results provide our new insights into the innate immune response during early infection by L. interrogans.

Klebsiella pneumoniae infection is one of the important reasons for the increased of morbidity and mortality. The main virulence factors of K. pneumoniae include capsule polysaccharide, lipopolysaccharide, fimbriae, outer membrane proteins and siderophores. BolA homologues form a broadly conserved family of proteins in prokaryotes and eukaryotes. In Escherichia coli, bolA expression is quickly induced in response to different stresses or stationary phase that rapidly adapt to changing environments. In this report, we confirmed that bolA mutant strain exhibited increased sensitivity to bile and oxidative stresses. In addition, gene deletion showed that bolA has an important role for the adherence of K. pneumoniae to host cell and establishment in mice, including liver, spleen, kidney and lung tissues, and induce the formation of liver abscess in mice. Our results also demonstrated that K. pneumoniae bolA increases the production of siderophore and virulence in Galleria mellonella larvae. Collectively, our results demonstrated that K. pneumoniae BolA is a new virulence factor which contributes to survival in different stresses and overcome host defense. These findings are helpful for the research of new treatment strategies for K. pneumoniae infection. IMPORTANCEKlebsiella pneumoniae is an important conditional pathogen causing nosocomial infections and community-acquired infections. It can resistant to multiple antibiotics, causing refractory infections and public health threat. Therefore, new treatments are required to fight the pathogen, and a better understanding of its virulence factors are needed to develop new drugs. Here, we unraveled the role of BolA in survival under different stresses and overcome host defense. Our results suggested that bolA actively contributes to cell morphology, stresses challenge, cell adhesion and siderophore production that are tightly related to bacterial virulence. Therefore, bolA mutant strain reduces the virulence of K. pneumoniae in G. mellonella larvae and its colonization ability in mice. These results reported bolA is a key virulence factor in K. pneumoniae, and they are helpful for research of new therapies to treat this increasingly problematic pathogen.

Salmonella enterica serovar Typhimurium causes acute diarrhea upon oral infection in humans. The harsh and proteolytic environment found in the gastrointestinal tract is the first obstacle that these bacteria face after infection. However, the mechanisms that allow Salmonella to survive the hostile conditions of the gut are poorly understood. The ecotin gene is found in an extensive range of known phyla of bacteria and it encodes a protein that has been shown to inhibit serine proteases. Thus, in the present work we studied the role of ecotin of Salmonella Typhimurium in host-pathogen interactions. We found that Salmonella Typhimurium {Delta}ecotin strain exhibited lower inflammation in a murine model of Salmonella induced colitis. The {Delta}ecotin mutant was more susceptible to the action of pancreatin and purified pancreatic elastase. In addition, the lack of ecotin led to impaired adhesion to Caco-2 and HT-29 cell lines, related to the proteolytic activity of brush border enzymes. Besides, {Delta}ecotin showed higher susceptibility to lysosomal proteolytic content and intracellular replication defects in macrophages. In addition, we found Ecotin to have a crucial role in Salmonella against the microbicide action of granules released and neutrophil extracellular traps from human polymorphonuclear leukocytes. Thus, the work presented here highlights the importance of ecotin in Salmonella as countermeasures against the host proteolytic defense system. IMPORTANCEThe gastrointestinal tract is a very complex and harsh environment. Salmonella is a successful food borne pathogen, but little is known about its capacity to survive against the proteolysis of the gut lumen and intracellular proteases. Here, we show that Ecotin, a serine protease inhibitor, plays an important role in protecting Salmonella against proteases present at different sites encountered during oral infection. Our results indicate that Ecotin is an important virulence factor in Salmonella, adding another tool to the wide range of features this pathogen uses during oral infection.