Virulence

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.

Polyamines are poly-cationic molecules ubiquitously present in all organisms. Salmonella synthesizes and also harbors specialized ABC transporters to uptake polyamines. Polyamines assist in pathogenesis and stress resistance in Salmonella; however, the mechanism remains elusive. The virulence trait of Salmonella depends on the injection of effector proteins into the host cell and modulation of host machinery and employs an array of arsenals to colonize in the host niche successfully. However, prior to this, Salmonella utilizes multiple surface structures to attach and adhere to the surface of the target cells. Our study solves the enigma of how polyamine spermidine assists in the pathogenesis of Salmonella. We show that spermidine mediates the initial attachment and adhesion of Salmonella Typhimurium to Caco-2 cells, facilitating its invasion. In-vivo studies showed that polyamines are required for invasion into the murine Peyers patches. Polyamines have previously been shown to regulate the transcription of multiple genes in both eukaryotes and prokaryotes. We show that spermidine controls the RNA expression of the two-component system, BarA/SirA, that further regulates multiple fimbrial and non-fimbrial adhesins in Salmonella. Flagella is also a vital surface structure aiding in motility and attachment to surfaces of host cells and gall stones. Spermidine regulated the expression of flagellin genes by enhancing the translation of s28, which features an unusual start codon and a poor Shine-Dalgarno sequence. Besides regulating the formation of the adhesive structures, spermidine tunes the expression of the Salmonella pathogenicity island-1 encoded genes. Thus, our study unravels a novel mechanism by which spermidine aids in the adhesion and the subsequent invasion of Salmonella into host cells.

Gastroenteritis causing pathogen Salmonella Typhimurium (S. Tm) during its infection in host cells thrives in a vacuolated compartment, Salmonella Containing Vacuole (SCV), which sequentially acquires host endosomal and lysosomal markers. Long tubular structures, called as Salmonella induced filaments (SIFs), are known to be required for SCVs nutrient acquisition, membrane maintenance and stability. A tightly coordinated interactions involving prominent effector SifA and various host adapters PLEKHM1, PLEKHM2 and Rab GTPases govern SCV integrity and SIF formation. Here, we report for the first time, the functional regulation of SifA is modulated by its SUMOylation at lysine 11. S. Tm expressing lysine 11 mutant SifA (SifAK11R) is defective in intracellular proliferation due to compromised SIF formation and enhanced lysosomal acidification. Furthermore, murine competitive index experiments reveal defective in vivo proliferation and weakened virulence of SifAK11R mutant. Concisely, our results demonstrate that SUMO deficient SifA mutant nearly behaves like a SifA knockout strain which impacts PLEKHM2-M6PR mediated lysosomal acidification pathway. Thus, our results bring forth a novel S. Tm-host crosstalk mechanism involving host mediated effector SUMOylation critical for pathogenicity.

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

Intracellular pathogens rely on manipulating host endocytic pathways to ensure survival. Legionella and Chlamydia exploit host SNARE proteins, with Legionella cleaving syntaxin 17 (STX17) and Chlamydia interacting with VAMP8 and VAMP7. Similarly, Salmonella targets the hosts endosomal fusion machinery, using SPI effectors like SipC and SipA to interact with syntaxin 6 (STX6) and syntaxin 8 (STX8), respectively, maintaining its vacuolar niche. Recent evidence highlights syntaxin 7 (STX7), a Qa-SNARE involved in endo-lysosomal fusion, as a potential Salmonella target. BioID screening revealed STX7 interactions with SPI-2 effectors SifA and SopD2, suggesting a critical role in Salmonella pathogenesis. We investigated the role of STX7 in Salmonella-containing vacuole (SCV) biogenesis and pathogenesis in macrophages and epithelial cells. Our findings indicate that STX7 levels and localization differ between these cell types during infection, reflecting the distinct survival strategies of Salmonella. Live cell imaging showed that STX7 is recruited to SCVs at different infection stages, with significantly altered distribution in HeLa cells at the late stage of infection. STX7 knockdown resulted in reduced bacterial survival, which was rescued upon overexpression of STX7 in both HeLa and RAW264.7 cells, suggesting Salmonella hijacks STX7 to evade lysosomal fusion and secure nutrients for intracellular replication. These results underscore the essential role of STX7 in maintaining SCVs and facilitating Salmonella survival. Further, the temporal expression of STX7 adaptor/binding partners in macrophages showed dynamic interactions with STX7 facilitating Salmonella infection and survival in host cells. Together, our study highlights STX7 as a critical host factor exploited by Salmonella, providing insights into the molecular mechanisms underlying its pathogenesis in macrophages and epithelial cells. These findings may in form strategies for targeting host-pathogen interactions to combat Salmonella infections.

Brucella species are Gram-negative intracellular bacterial pathogens that cause the worldwide zoonotic disease brucellosis. Brucella can infect many mammals, including humans and domestic and wild animals. Brucella manipulates various host cellular processes to invade and multiply in professional and non-professional phagocytic cells. However, the host targets and their modulation by Brucella to facilitate the infection process remain obscure. Here, we report that the host Ubiquitin Specific Protease, USP8 negatively regulates the invasion of Brucella into macrophages through the plasma membrane receptor, CXCR4. Brucella suppressed the expression of USP8 at its early stage of infection in the infected macrophages. Subsequent studies revealed that the Brucella effector protein, TIR-domain containing protein from Brucella, TcpB plays a significant role in downregulating the expression of USP8 by targeting the CREB pathway. Treatment of mice with USP8 inhibitor resulted in enhanced survival of B. melitensis, whereas mice treated with CXCR4 or 14-3-3 antagonist showed a diminished bacterial load. Our experimental data demonstrate a novel role of USP8 in the host defence against microbial intrusion and microbial subversion of host defences.

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.

Shiga toxing-producing Escherichia coli (STEC) O22:H8 strain is a serotype occasionally isolated in Argentinian cattle. Preliminary works showed that the cattle carrying STEC O22:H8 strains could not be experimentally colonized by EHEC O157:H7 strain. The type 6 secretion system (T6SS) is one of the most versatile virulence mechanisms involved in delivering effectors, particularly the T6SS1 translocate antibacterial toxins effectors during bacterial competition for niche-space. In this work, we could evidence the molecular bases of the success of STEC O22:H8 (154) strain during bacterial competition against EHEC O157:H7 strains. The genome sequence of STEC O22:H8 (154) allowed us to identify a complete T6SS1 cluster. In addition, we identify and characterized several putative T6SS1-antibacterial effectors encoded inside the T6SS1 clusters and in genomic pathogenic islands. Competition assays against EHEC O157:H7 strain confirmed the antibacterial activity of STEC O22:H8 (154) strain in vitro. Considering the absent of T6SS1 in STEC strains, we proposed the recent horizontal transfer acquisition and the most probably donor belong to the same Escherichia coli species. A safe STEC O22:H8 (154){Delta} stx would be used as a new strategy to fight STECs in bovine intestinal colonization, leading in a reduction in beef contamination and consequently HUS cases in humans.

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.

ObjectivesInvestigating the type 1-E CRISPR-Cas-mediated regulation of Salmonella pathogenesis. MethodsWe assessed the pathogenicity of the wild-type and CRISPR-Cas knockout strains using infection models. The mechanisms were elucidated using antimicrobial assays and expression analysis. ResultsCRISPR-Cas knockout strains were defective in invasion and proliferation in intestinal epithelial cells and macrophages. However, proliferation defects were not observed in the Gp91phox-/- macrophages, suggesting the systems role in antioxidant defence. The knockout strains show hampered colonization in in-vivo infection models, possibly due to increased sensitivity against innate immune barriers like antimicrobial peptides, complement proteins and oxidative stress. The expression studies of various virulence regulators: pmr genes, anti-oxidant genes, SPI-1 and SPI-2 encoded master regulators, and effectors showed repressed expression in the knockout strains. Some of these genes could be directly regulated by the CRISPR-spacers owing to partial complementarity between the sequences. ConclusionOverall, our study shows that the CRISPR-Cas system positively regulates Salmonella pathogenesis by regulating the expression of different virulence factors.

Salmonella infection involves a cascade of attacks and defence measures. After breaching the intestinal epithelial barrier, Salmonella is phagocytosed by the macrophages, inside which, the bacteria face multiple stresses and, consequently, employ appropriate countermeasures. We show that, in Salmonella, the polyamine spermidine activates a stress response mechanism by regulating critical antioxidant genes. Salmonella Typhimurium mutants for spermidine transport and synthesis cannot mount an antioxidative response, resulting in high intracellular ROS levels. These mutants are also compromised in their ability to be phagocytosed by macrophages. Furthermore, it regulates a novel enzyme in Salmonella, Glutathionyl-spermidine synthetase (GspSA), which is known to prevent the oxidation of proteins in E.coli. Moreover, the spermidine mutants and the GspSA mutant show significantly reduced survival in the presence of hydrogen peroxide in vitro, and lesser organ burden in the mouse model of Salmonella infection. Conversely, in macrophages isolated from gp91phox-/- mice, we observed a rescue in the attenuated fold proliferation previously observed upon infection. Interestingly, Salmonella upregulates polyamine biosynthesis in the host through its effectors from SPI-1 and SPI-2, which also solves the mystery of the attenuated proliferation observed in spermidine transport mutants. Thus, inhibition of this pathway in the host abrogates the proliferation of Salmonella Typhimurium in macrophages. From a therapeutic perspective, inhibiting host polyamine biosynthesis using an FDA-approved chemopreventive drug, D,L--difluoromethylornithine (DFMO), reduces Salmonella colonization and tissue damage in the mouse model of infection, while enhancing the survival of infected mice. Therefore, our work provides a mechanistic insight into the critical role of spermidine in stress resistance of Salmonella. It also reveals a strategy of the bacteria in modulating host metabolism to promote their intracellular survival and shows the potential of DFMO to curb Salmonella infection.

Coxiella burnetii is an obligate intracellular zoonotic bacterium that causes Q fever. Infections can be either acute or chronic. Of note, chronic Q fever develops months or years after primary infection without clinical symptoms, suggesting bacterial persistence. Yet, information about the induction, regulation and/or location of C. burnetii persistence is rare. We have shown that during infection of primary macrophages, hypoxia-induced citrate limitation results in inhibition of C. burnetii replication without affecting viability. Here, primary murine macrophages were infected with C. burnetii under normoxic (21% O2) and hypoxic (0.5% O2) conditions to clarify how C. burnetii survives this environmental stress condition. Our data suggests that under hypoxic conditions C. burnetii does not undergo stringent response, but instead enters a SCV-like form, which is smaller in size and possesses condensed chromatin material and a thicker cell wall. These changes have functional consequences, as the SCV-like persistent form of C. burnetii is more infectious, more tolerant to antibiotics and less sensitive to clearance by IFN{gamma} activated macrophages. Hence, the development of the SCV-like persistent form of C. burnetii prevents elimination of the pathogen, which in turn allows the pathogen to thrive once the conditions again change in its favor.

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

After entering the host cells, Salmonella Typhimurium (STM) stays inside a modified membrane-bound compartment called Salmonella containing vacuole (SCV). The biogenesis and stability of SCV are crucial for the intracellular proliferation of Salmonella. Our research has provided a novel mechanistic view on the role of a bacterial porin OmpA in maintaining the stability of SCV. We found that the deletion of OmpA forces the bacteria to escape from the SCV during the immediate early stage of infection. In the absence of OmpA, the bacteria failed to retain the LAMP-1 and came into the host cells cytosol. Subsequently, the cytosolic population of STM{Delta} ompA activated the host autophagy machinery after colocalizing with syntaxin 17 and LC3B. The autophagosomes carrying STM{Delta} ompA were targeted to the lysosomes for degradation. Inhibition of autophagy pathway using bafilomycin A1 restored the intracellular proliferation of STM{Delta} ompA. We further showed that the four extracellular loops of OmpA played a crucial role in holding the LAMP-1 pool around the SCV. We have altered the extracellular loop sequences of Salmonella OmpA by site-directed mutagenesis and observed that the bacteria failed to maintain the LAMP-1 pool around the SCV, which finally resulted in their release into the cytosol of the host macrophages. Surprisingly, the cytosolic population of Salmonella having mutations in the extracellular loops of OmpA didnt activate the lysosomal degradation pathway like STM{Delta} ompA, which helped them to survive within the murine macrophages. In summary, our study revealed an OmpA dependent novel strategy utilized by Salmonella to combat host autophagy by promoting the stability of SCV.

The Chlamydiota phylum consists of obligate intracellular bacteria, including well-known pathogens and emerging environmental species, with diverse host ranges and metabolic capabilities. Among these bacteria, the gene, which encodes nucleoside diphosphate kinase (ndk), is present in variable copy numbers. While most chlamydial species carry a single copy of ndk, some species have two copies. In W. chondrophila, the two Ndk proteins encoded by ndk paralogs retain conserved kinase motifs but differ in subcellular localization, suggesting divergent functional roles. According to localization studies performed in heterologous expression systems, WcNdk1 is confined to the inclusion and probably supports nucleotide metabolism, while WcNdk2 localizes to the host nucleus, perinuclear space, and Golgi apparatus, suggesting involvement in host interaction. Azidothymidine (AZT), a known Ndk inhibitor, impaired W. chondrophila growth, potentially through inhibition of WcNdk2. However, the lack of genetic tools and the absence of in vitro enzymatic assays currently limit definitive functional conclusions. Our data suggest potential functions for Ndks in W. chondrophila, providing a foundation for future studies on Ndk-mediated interactions between this pathogen and its host.

The upregulation of PD-L1 by various pathogens is a recognized strategy to evade the adaptive immune response. Salmonella infection also upregulates PD-L1 levels causing culling of the activated T-cell; however, the underlying mechanism behind this upregulation is not known. Our findings indicate that the upregulation of PD-L1 is through Salmonella pathogenicity island 2 (SPI-2) encoded effectors since PFA-fixed STM WT and STM{Delta}ssaV (which is unable to secrete effector proteins) did not alter PD-L1 levels. We have further investigated the role of the SPI-2 effector SseL (a deubiquitinase known to affect the NF-B pathway) in PD-L1 upregulation. Our study identifies SPI-2 effector SseL to be crucial for upregulating PD-L1 in vitro as well as in vivo murine models. The increase in PD-L1 levels induced by STM WT facilitates colonization in secondary infection sites in C57BL/6 mice, including the liver and spleen, while the STM{Delta}sseL strain exhibits significant colonization defects. Notably, despite the reduced colonization capacity of STM{Delta}sseL, infected mice exhibit earlier mortality associated with heightened inflammation. We further elucidated the molecular mechanism behind PD-L1 upregulation and observed that bacterial effector SseL helps in the stabilization of {beta}-catenin inside the cell. {beta}-catenin thus translocates into the nucleus and directly regulates the transcriptional levels of PD-L1, which is abrogated upon using {beta}-catenin/TCF inhibitor FH535. Collectively, our study elucidates the mechanism by which Salmonella mediates immune suppression through PD-L1 upregulation. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/620790v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@12ba486org.highwire.dtl.DTLVardef@3e8b40org.highwire.dtl.DTLVardef@28546corg.highwire.dtl.DTLVardef@1bea897_HPS_FORMAT_FIGEXP M_FIG Abstract figure: Schematic representation of SseL mediated PDL1 upregulation and further affecting the T cell proliferation C_FIG

An opportunistic fungal pathogen, Cryptococcus neoformans (C. neoformans), causes cryptococcal meningitis and is frequently associated with high mortality in immunocompromised individuals, particularly in HIV patients. Formation of metabolically altered lipid-rich foamy macrophages has been reported as a successful strategy employed by various intracellular pathogens to secure a nutrient source and niche within the host. Herein, we elucidate the involvement of macroautophagy, specifically lipophagy, in lipid dysregulation during C. neoformans infection. Our study highlights a pivotal role of lipophagy during infection, showing that C. neoformans driven activation of WNT-signaling leads to an aberrant lipid accumulation in host macrophages under the regulatory role of a histone modifier, Lysine Specific Demethylase 1 (LSD1). In a murine model of pulmonary infection, targeting host LSD1 led to a significant reduction in lung fungal burden, accompanied by amelioration of lung pathology and reduction of lipid content in the lungs. The study highlights the significance of host epigenetic regulation in modulating foamy macrophage formation through the regulation of lipophagy during C. neoformans pathogenesis.

GBS may cause a devasting disease in newborns. In early onset disease of the newborn the bacteria are acquired from the colonized mother during delivery. We characterized type VII secretion system (T7SS), exporting small proteins of the WXG100 superfamily, in group B Streptococci (GBS) isolates from pregnant colonized women and newborns with early onset disease (EOD) to understand better understand T7SS contribution to virulence in these different clinical scenarios. GBS isolates were obtained from colonized mother prior to delivery and from newborns with EOD. DNA was analyzed for T7SS genes. A mutant EOD strain (ST17) was created by knocking out the essC gene encoding a T7SS protein. Galleria mellonella larvae were used to compare virulence of colonizing, EOD, and mutant EOD isolates. 33 GBS genomes were tested, 17 EOD isolates and 16 colonizing isolates. The T7SS locus encoded 8 genes: essC, membrane-embedded proteins (essA; essB), modulators of T7SS activity (esaA; esaB; esaC) and effectors: [esxA (SAG1039); esxB (SAG1030). ST17 isolates encode two copies of the essC gene and esxA gene encoding putative effectors but were present only in 23.5% of isolates. In ST1 isolates three copies of esxA gene were identified, but in ST6 and ST19 isolates all T7SS genes were missing. EOD isolates demonstrated enhanced virulence in G. mellonella model compared to colonizing isolates. The 118659{Delta}essC strain was attenuated in its killing ability, and the larvae were more effective in eradicating 118659{Delta}essC infection. essC gene deletion was associated with reduced bacterial growth. We demonstrated that T7SS plays an essential role during infection and contributes to GBS pathogenicity. Author SummaryType VII secretion system (T7SS) is related to virulence in various bacteria but is not well characterized in Group B Streptococci (GBS). GBS may cause sepsis, meningitis, and death in newborns. The bacteria rarely cause disease in pregnant mothers. Newborns acquire GBS from the colonized mother during delivery. We studied the role of T7SS in GBS isolates obtained from newborns with GBS sepsis in the first week of life and in colonized pregnant mothers. By studying T7SS genes we discovered that the genetic structure of the T7SS differs between isolates causing severe disease and colonizing isolates. To study the virulence of different GBS isolates we injected them into larvae and monitored larvae survival. Isolates causing severe disease in the newborn caused a more severe disease in larvae compared to colonizing isolates. We then deleted T7SS genes in GBS isolates causing severe disease. The killing activity of GBS isolates without T7SS genes was attenuated. The larva responded to these bacteria similarly to the response found when injecting the larva with GBS isolates from colonized mothers. These results support our hypothesis that T7SS is important for causing severe infection in the newborn and that this system contributes to GBS pathogenicity.

Encephalitozoon cuniculi is an opportunistic intracellular pathogen that establishes a balanced relationship with immunocompetent individuals depending on the activity of their CD8+ T cells lymphocytes. However, lower resistance to experimental infection with E. cuniculi was found in B-1 deficient mice (Xid), besides increased the number of CD8 T lymphocytes. Here, we evaluated the cytotoxic activity of CD8+ T lymphocytes from Balb/c wild-type (WT) or Balb/c Xid mice (with B-1 cell deficiency) on the microbicidal activity of macrophages challenged with E. cuniculi. CD8 T lymphocytes from WT or Xid mice previously infected or not with E cuniculi were co-cultured with macrophages challenged with E. cuniculi. We evaluated macrophages viability and microbicidal activity, and proliferation, viability, and presence of activating molecules (CD62L, CD69, and CD107a) in CD8 T lymphocytes. Macrophages co-cultured with CD8 T lymphocytes from WT demonstrated high microbicidal activity. CD8 T lymphocytes obtained from uninfected WT mice had a higher proliferative capacity and a higher expression of CD69 and LAMP-1-activating molecules compared to Xid CD8+ T lymphocytes. CD8 T lymphocytes from infected Xid mice proliferated more than those from WT mice, however, when the expression of the activating molecule CD69 associated with the expression of CD62L was kept low. In conclusion, the absence of B-1 cells in Xid mice might be associated with the lower expression of activating molecules in CD8+ T lymphocytes and their cytotoxic activity. However, after a previous infection with E. cuniculi, CD8 T lymphocytes were more effective in killing macrophages infected with E. cuniculi.

Antibiotic resistance of pathogenic bacteria has emerged as a major threat to public health worldwide. While stable resistance due to the acquisition of genomic mutations or plasmids carrying antibiotic-resistance genes is well-established, much less is known about the temporary and reversible resistance induced by antibiotic treatment, such as the one due to treatment with bacterial cell-wall inhibiting antibiotics like ampicillin. Typically, ampicillin concentration in the blood and other tissues gradually increases over time after initiation of the treatment. As a result, the bacterial population is exposed to a concentration gradient of ampicillin. This is different from in vitro drug testing where the organism is exposed to fixed drug concentrations from the beginning till the end. To mimic the mode of antibiotic exposure of microorganisms in the tissues, we cultured the wild type, ampicillin-sensitive Salmonella Typhi Ty2 strain (S. Typhi Ty2) in the presence of increasing concentrations of ampicillin over a period of 14 days. This resulted in the development of a strain that exhibited several features of the so-called L-form of bacteria, such as the absence of cell wall, altered shape and slower growth rate compared with the parental strain. Studies on the pathogenesis of S. Typhi L-form showed efficient infection of the murine and human macrophage cell lines. More importantly, S. Typhi L-form was also able to establish infection in a mouse model to the extent comparable to its parental strain. These results suggested that L-form generation following initiation of antibiotic treatment could lead to drug escape of S. Typhi and direct spread to new cells (macrophages), which sustain the infection. Oral infection by the L-form bacteria underscores the potential of rapid disease transmission through faeco-oral route, highlighting the need for new approaches to decrease the reservoir of infection.