Virulence

BackgroundInfectious bovine keratoconjunctivitis is the most important cattle ocular disease worldwide. The infection is primarily caused by Moraxella bovis and is a highly contagious disease that significantly affects cattle welfare. Currently, antibiotic medication is the primary treatment for infectious bovine keratoconjunctivitis. However, with rising concerns over antibiotic resistance, we propose developing a more targeted therapeutic strategy using bacteriophages (phages). Materials and MethodsWe have isolated the first known Moraxella bovis phages, characterised them according to their genome sequence, local virulence index and with transmission electron microscopy. The host ranges were assessed using 41 clinical M. bovis strains isolated from infected cows. ResultsFour phages were isolated and characterised. Comparative analysis identified a high degree of genomic similarity between the phages MB15, MB16, MB26 and MB43. MB43 was the most distinct, with the smallest host range phenotype. ConclusionsThe isolated phages show therapeutic potential for further development against Moraxella infections.

Orientia tsutsugamushi is a mite-transmitted obligate intracellular bacterium that causes the potentially deadly zoonosis, scrub typhus. The absence of genetic tools for Orientia have limited studies of the microbe-host interactions that underlie scrub typhus. To address this gap, we developed a protocol for transforming and achieving allelic exchange in O. tsutsugamushi str. Ikeda. From evaluating multiple cell lines and antibiotics, we found that contact-inhibited EA.hy926 human endothelial-like cells best supported Orientia replication and that chloramphenicol was an effective selection marker. We engineered a homologous recombination cassette encoding a codon-modified version of the O. tsutsugamushi ank13 gene (OTT_RS04140) (CMank13) and its promoter alongside genes for mScarlet-I and chloramphenicol acetyltransferase under control of the O. tsutsugamushi tsa22-up and tsa56-down promoters, respectively. A PCR product encompassing the cassette and chromosomal flanking regions was transformed into O. tsutsugamushi via electroporation or CaCl2, the latter of which better preserved bacterial and host cell viability. EA.hy926 cells inoculated with transformed O. tsutsugamushi were grown in glass-bottom plates in the presence of chloramphenicol and imaged by live-cell microscopy to identify cultures containing mScarlet-I positive bacteria, which could be maintained in perpetuity. Chromosomal integration of the CMank13 cassette and loss of wild-type ank13 were verified by PCR and nanopore sequencing. This report establishes platforms for genetically manipulating O. tsutsugamushi and building additional genetic tools to investigate this globally significant pathogen. IMPORTANCEOrientia tsutsugamushi causes scrub typhus, a globally emerging rickettsiosis that can have a high mortality rate and has been a known human disease since the fourth century. Of the genera of obligate intracellular bacterial pathogens that cause human disease, Orientia is the only one for which genetic tools have not been developed. This has limited understanding of O. tsutsugamushi-host dynamics that drive the bacteriums pathobiology and hindered development of novel treatment or protection strategies against scrub typhus. Here, we successfully transformed and achieved allelic exchange in O. tsutsugamushi. Transgenic bacteria were selected via antibiotic resistance, validated by PCR and nanopore sequencing, and visualized by immunofluorescence and live-cell fluorescence imaging. Our report includes detailed descriptions of empirically determined host cell cultivation, multiplicity of infection, transformation, and selection conditions to provide a foundation on which other researchers can build. Overall, this work begins to establish a genetic toolbox for O. tsutsugamushi.

The pathogenic fungus Trichosporon asahii causes severe mycoses in immunocompromised hosts, such as neutropenic patients. In Cryptococcus neoformans, the unfolded protein response (UPR) sensor Ire1 induces hxl1 mRNA splicing and contributes to stress responses and virulence. The function of Ire1-triggered hxl1 mRNA splicing in stress tolerance and virulence of T. asahii, however, remains unclear. Here, we demonstrated that ire1- and hxl1 gene-deficient T. asahii mutants are sensitive to dithiothreitol (DTT), an inducer of endoplasmic reticulum stress, and exhibit reduced virulence in a silkworm infection model. DTT treatment induced hxl1 mRNA splicing in the wild-type strain, whereas ire1 gene-deficient mutants did not undergo hxl1 mRNA splicing. The ire1 gene-deficient mutants were more sensitive than the parent strain to DTT, H2O2, Congo red, and SDS, and showed reduced virulence in silkworms. Similarly, hxl1 gene-deficient mutants exhibited increased sensitivity to these stressors and reduced virulence. Both the ire1 gene-deficient and hxl1 gene-deficient mutants showed decreased expression of reactive oxygen species-detoxifying related genes CAT2, SOD1, and SOD2, compared with the parent strain. Together, these findings suggest that Ire1-triggered hxl1 mRNA splicing contributes to stress resistance and virulence in T. asahii.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that undergoes lytic replication in epithelial and other permissive cell types and can establish latency in peripheral neurons. ICP22 is a multifunctional HSV-1 immediate-early protein that localizes to the nucleus of infected cells; however, its interactions with host cellular factors remain incompletely understood. Here, ICP22 was demonstrated to interact with the human antisense function 1 protein (ASF1), including both ASF1a and ASF1b, in transfected cells and HSV-1-infected cells, respectively. ICP22 also colocalized with ASF1 in the nucleus. ICP22 amino acids 213 to 340 are important for the interaction of ICP22 with ASF1, whereas amino acids 37 to 153 of ASF1a and ASF1b are critical for their interactions with ICP22. Furthermore, ICP22 expression was associated with reduced ASF1-H3.1 co-immunoprecipitation under the tested conditions. ASF1 knockdown also reduced HSV-1-BAC-Luc luciferase output, indicating that ASF1 contributes to efficient infection-associated reporter activity in this study. Collectively, these results indicate that the interaction of HSV-1 ICP22 with ASF1 might help regulate the transcription of viral or cellular genes during HSV-1 infection. Keywords: HSV-1, ICP22, ASF1, histone H3.

BackgroundStaphylococcus aureus (S. aureus) is an increasingly recognized intracellular pathogen, yet infection outcomes vary with bacterial isolate and host cell type. The mechanisms underlying these differences remain poorly understood. This study investigates how distinct intracellular S. aureus isolates influence host signaling programs and infection outcomes by modulating cell death pathways and TNF-R1 dependent regulation of host cell fates across different human cell lines. MethodsFour S. aureus isolates were analyzed for intracellular localization using transmission electron microscopy (TEM), structured illumination microscopy (SIM), serial block-face scanning electron microscopy (SBF-SEM), and imaging flow cytometry. Transcriptional reprogramming of infected U937 monocytes was examined by mRNA sequencing. Infection outcomes were characterized and compared to A549 and SaOS-2 cell lines employing Luminex cytokine assays, flow cytometry and Western blot analysis to characterize host cell death mechanisms in both wild-type and TNF-R1 deficient backgrounds. ResultsAll S. aureus isolates localized to endolysosomal and cytosolic compartments but also peri and putatively intranuclearly, revealing an unexpected intracellular niche. In U937 monocytes, infection induced a conserved stress signature alongside isolatespecific transcriptional programs divergently affecting inflammation, metabolism, and cell fate, which was markedly attenuated in response to the chronicinfection isolate EDCC 5464. Cell death outcomes were likewise isolatedependent, involving intrinsic and extrinsic apoptosis, mitochondrial depolarization, and caspase-1 activation at distinct temporal dynamics. TNFR1 loss initially delayed but exacerbated late, isolate-independent cytotoxicity, identifying TNFR1 as a key regulator of U937 infection outcome. SaOS2 and A549 cell death was far less affected by isolate or TNF-R1 deficiency. ConclusionsThese results highlight the multilayered determinants governing intracellular S. aureus survival, non-canonical intracellular localization, and host cell susceptibility. The TNF/TNF-R1 axis is identified to critically determine regulated host defense during early infection stages in a tissue-specific manner. Together with distinct isolate-driven gene expression profiles, infection risks under TNF-targeted therapies and the contribution of S. aureus heterogeneity should be considered in the design of future host-directed treatment strategies. Plain English summaryThe bacterium Staphylococcus aureus (S. aureus) often lives harmlessly in humans but can cause severe or recurrent infections when the skin barrier is broken or the immune system is weakened. A major reason for its persistence is its ability to hide inside human cells, where it is shielded from immune attacks and antibiotics. To effectively target such bacteria, it is crucial to understand that infections vary depending on both the bacterial strain and the infected cell type. Many reasons behind these differences are still puzzling. We explored how different types of S. aureus (collected from different disease types) change how human cells respond to infection. We focused on how the different strains influence the way immune cells adjust their gene activity during infection, and how a receptor called TNF-R1 is involved in managing cell death responses. Bacteria were found not only in compartments meant to destroy them but also near and even inside the cell nucleus, an unexpected location. All strains triggered a similar stress response but also distinct patterns influencing inflammation, metabolism, and cell survival. A strain linked to chronic infection caused weaker responses, suggesting greater stealth. Cells lacking TNF-R1 initially survived longer but later showed greater damage, indicating this receptors role in infection control. In lung and bone cells, these effects were less pronounced. Concludingly, S. aureus occupies unexpected niches inside human cells and uses varying survival strategies. TNF-R1 is a key regulator of host infection responses in the analyzed immune cells, highlighting that both bacterial diversity and host factors must be considered when developing targeted treatments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/723175v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1b4214org.highwire.dtl.DTLVardef@18f4ee6org.highwire.dtl.DTLVardef@1851742org.highwire.dtl.DTLVardef@ba0359_HPS_FORMAT_FIGEXP M_FIG Peri- and intranuclear localization early after S. aureus uptake across host cell lines, with isolate-specific modulation of host fates and a critical role for TNF-R1 to mediate regulated death responses of U937 cells. At 2 hpi, intracellular S. aureus not only localizes in (LAMP-1 decorated) membrane-enclosed compartments or directly in the cytosol, but within invaginations of the nuclear surface and intranuclearly with or without being surrounded by a vesicular membrane in U937wt, SaOS-2wt, and A549wt cells. At 4 hpi, S. aureus triggers differential gene expression in (A) U937wt cells to an isolate-specific extent, with both unique and shared transcriptomic signatures across the four isolates, that is muted for the chronic infection isolate EDCC 5464. Apoptotic cell death is induced to an isolate-dependent extent involving extrinsic initiator caspase-8, intrinsic initiator caspase-9 (EDCC 5055 only), and variable effector caspase-3/-7 activity in the earlier stages of infection (6 hpi), which then barely increases (24 hpi) in U937wt cells. S. aureus-induced cell death and caspase activation is abolished in (B) U937{Delta}TNF-R1 at 6 hpi, but is significantly reinforced at 24 hpi with diminished isolate-specificity. Correspondingly, mitochondrial trans-membrane potential ({Delta}{Psi}m) is disrupted for all isolates upon TNF-R1 knockout, as well as caspase-1 activity, suggesting pyroptotic pathway activation at later stages of infection. (C) SaOS-2 wt cells show moderate caspase-3/-7 and -1 activation, while infection induces detachment of (D) A549wt cells with minimal caspase activation. Infection induces an isolate- and cell line-dependent cytokine release. Coloured arrows indicate the mean proportion of effector-positive cells ({uparrow} [~]20-40%, {uparrow} {uparrow} 40-60%, {uparrow} {uparrow} {uparrow} >60%) representing each S. aureus isolate. Grayed signaling arrows indicate the hypothesis by which TNF-R1 activation and internalization is required to kill lysosomal S. aureus via activation of anti-microbial enzymes and downstream regulated death pathway activation. Created with BioRender.com. C_FIG

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating immune responses and have emerged as potential biomarkers and therapeutic targets in complex diseases. Leishmaniasis is a neglected disease that compromises host immunity and is associated with challenging treatments regimens. Leishmania amazonensis (L. amazonensis), an intracellular protozoan parasite, causes cutaneous leishmaniasis by replicating inside mammalian macrophages to establish infection. In this context, miRNAs have emerged as vital post-transcriptional factors that regulate the inflammatory landscape during infection. In this study, we aimed to analyze the function of miR-721 in macrophages during L. amazonensis infection by integrating in silico miR-721 target prediction with RNAseq data from macrophages of two distinct mouse genotypes, resistant C57BL/6 and susceptible BALB/c. We found that miR-721 is induced in macrophages infected with L. amazonensis, but is not in LPS-stimulated macrophages, suggesting a TLR4-independent activation. Integrating miR-721 target prediction with comparative transcriptomic analyses in resistant C57BL/6 and susceptible BALB/c models revealed the TNF-IRF1 axis as a primary miR-721-associated regulatory network. Specifically, miR-721 is predicted to target the 3UTRs of Tnf and Irf1 to suppress the inflammatory response. Functional inhibition of miR-721 successfully restored Tnf and Irf1 expression and reduced the amastigote burden over 24 hours. Furthermore, we showed that the miR-721/TNF-IRF1 axis regulates downstream genes associated with macrophage response, such as Serpine1, Csf1, Cd69 and Maf. Our work demonstrated that Leishmania induces miR-721, which negatively modulates the TNF-IRF1 axis, thereby suppressing the immune response and favoring parasite persistence. While C57BL/6 macrophages exhibit a robust activation of the TNF-IRF1 network, promoting inflammatory response, BALB/c macrophage showed a breakdown of this network. This was associated with post-transcriptional suppression of inflammatory responses, thereby favoring parasite persistence. These findings link miR-721 to the establishment of macrophage polarization, providing relevant insights into the mechanisms of parasite subversion of the host immune response.

Streptococcal pyrogenic exotoxin C (SpeC) is a prototypical superantigen produced by group A Streptococcus. It potently activates a broad subset of T lymphocytes via a bridging interaction involving TCR{beta}-SpeC-MHC-II. Our recent work demonstrated that SpeC induced profound release of IL-8 from human pharyngeal epithelial cells and this effect was reversible through a specific point mutation in SpeC. This study systematically investigated cellular signaling pathways using integrated transcriptomic profiling and Western blot analysis, with a focus on membrane-associated receptors and downstream intracellular signaling effectors. Our results demonstrate that this biological process is critically associated with the activation of Erk1/2, p38 MAPK and NF-{kappa}B signaling cascade. This study identifies a novel mechanism through which a bacterial superantigen target epithelial cells-the body primary physical barrier and first line of innate immune defense.

The migratory endoparasitic pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, causing significant economic and ecological losses in conifer forest ecosystems in Europe and Asia. Understanding the molecular mechanisms regulating PWN parasitism-related genes may lead to new sustainable solutions for control. Based on previous PWN transcriptomic datasets from the pre-parasitic and parasitic stages and from the pharyngeal gland cells (GC), an in silico analysis was performed to identify transcription factors (TF) highly expressed in the GC. Seven candidates TF genes were selected, and their spatial expression validated by in situ hybridisation. From those, two GC-expressed TFs, BXY_079 and BXY_022, each encoding zinc finger domains, were successfully knocked down by RNA interference. Transcriptomic data from silenced BXY_079 and BXY_022 TFs, analysed with existing life cycle specific transcriptomic data, showed that both TFs control genes expressed at similar times, by repressing male-related genes while activating genes expressed during the J3 and D3 stages, yet each represents the extreme of the others minor function. In addition to these common roles, BXY_079 also activates parasitism-related genes in the J2 stage. These BXY_079-activated parasitism-related genes predominantly encode proteins with lytic functions, including secreted peptidases and glycoside hydrolases. Consistent with their proposed role in parasitism, these genes are highly expressed during the parasitic juvenile stages and are likely involved in nematode feeding, tissue penetration, and migration within the host. In contrast, BXY_022 also represses the expression of several genes related to the reproduction system, such as major sperm proteins and cytosolic motility proteins, particularly in the adult male stage. Taken together, both dual-functional TFs work together, non-redundantly, to regulate gene expression across the life cycle, while each is additionally specialised to regulate diverse and distinct gene sets: ranging from genes implicated in lytic parasitic functions to sexual dimorphism.

Treponema pallidum ssp. pallidum, the causative agent of syphilis, has a small proteome and encompasses numerous strains. Knowledge gaps remain in understanding the molecular mechanisms of pathogenesis of this bacterium, as well as the structure and function of the full complement of proteins encoded by T. pallidum. Here, an AI-based structure-to-function modeling workflow was used to investigate the complement of proteins encoded by T. pallidum. High-confidence structure models were generated for 976 T. pallidum proteins, covering 99% of the proteome. Analysis of the generated models using the protein structure comparison server DALI enabled high-confidence, structure-based functional annotation of 877 T. pallidum proteins, including 240 of the 323 proteins of unknown function encoded by this pathogen. Additionally, 63 putative pathogenesis related proteins (PPRPs) and seven treponemal proteins with previously uncharacterized similarity to outer membrane proteins (OMPs) from Gram-negative bacteria were identified. A workflow for B cell epitope (BCE) prediction identified 1133 surface-exposed, host-facing potential epitopes in known and predicted T. pallidum OMPs, of which 92 were prioritized based on bioinformatic analyses, biophysical properties, amino acid sequence conservation, and previous protein expression data. This work provides insight into T. pallidum pathogenesis through structure modeling-based functional annotation, including characterization of proteins of unknown function. This study also informs syphilis vaccine design by identifying new potential T. pallidum OMPs, as well as host-facing regions of T. pallidum OMPs that have conserved amino acid sequences in globally circulating strains. Statement of importance/impactThis study presents the first AI-based global structure modeling-to-function analysis of the proteome of Treponema pallidum, the bacterium that causes syphilis. Structure-based functional predictions of previously uncharacterized proteins, including proteins potentially involved in virulence, provide novel insight into mechanisms of pathogenesis. The work also informs syphilis vaccine development by the identification and structural characterization of new candidate vaccine proteins in globally circulating strains of T. pallidum.

MERS-CoV poses a constant pandemic risk, as its viral lineages continue evolving, and zoonotic spillover events could lead to random viral polymorphisms that might lead to human adapted variants. Currently, no small animal model reliably recapitulates both disease progression and transmission dynamics, which are critical aspects for counter-viral measures like vaccine development. Although the Syrian hamster is an optimal animal model for SARS-CoV-2 infection and transmission, it is naturally resistant to MERS-CoV infection. Dipeptidyl peptidase-4 (DPP4) is the functional receptor for MERS-CoV infection, and is highly expressed in human kidney, intestine, liver, and lung tissues. Here, we evaluated the suitability of a human DPP4 (hDPP4) transgenic Syrian hamster model for MERS-CoV research. We used two different MERS-CoV strains (EMC/2012 and D10540/2023) for intranasal inoculation of hamsters. Both strains replicated efficiently, led to comparable severe clinical outcomes, and had similar viral transmission efficiencies. MERS-CoV RNA and nucleoprotein antigen were mainly detected in the brain and the respiratory tract. In summary, we validated a novel hDPP4-transgenic hamster as a suitable model for MERS-CoV infection enabling vaccine and transmission research.

The IFN-I (type I interferon) signaling pathway is the first line of defence against foreign pathogens. Stringent control of signalling pathways is necessary to maintain host immune responses and homeostasis. However, the underlying mechanism for its tight regulation is yet completely understood. In this study, we demonstrated that the TRIM family protein tripartite motif-containing 52 (TRIM52) is a novel negative regulator of IFN-{beta} production. Ectopically expressed TRIM52 markedly inhibited the activation of the IFN-{beta} promoter by ectopic expression of cGAS/STING, RIG-IN, or TRIF, MAVS, STING, and TBK1 but not by IRF3/5D, indicating that TRIM52 targets TBK1. TRIM52 also significantly inhibited the IFN-{beta}, ISG54, and ISG56 production, the dimerization of IRF3 and the nuclear localization of IRF3-YFP induced by ectopic expression of TBK1. Co-immunoprecipitation experiment revealed that TRIM52 specifically interacted with TBK1. Furthermore, the TBK1 protein, but not its mRNA, decreased considerably with increasing expression of TRIM52, and TRIM52 did not decrease the expression of the cGAS, STING, or IRF3 proteins. In addition, proteasome inhibitor MG-132 blocked the reduced TBK1 induced by TRIM52, indicating that TRIM52 caused TBK1 degradation via the proteasome pathway. Co-IP and ubiquitination assays demonstrated that TRIM52 promotion of K48-linked ubiquitination of TBK1, which depends on its E3 ubiquitin ligase. Collectively, our findings identify a previously unrecognized role of TRIM52 in regulating the IFN-I signalling pathway through targeting TBK1 for polyubiquitination and degradation.

Shigella sonnei is a leading cause of bacterial dysentery and a high priority WHO pathogen because of the spread of multidrug resistant strains. Understanding microbiome-Shigella-host interactions during colonization of the gastrointestinal tract, and the development of vaccines have been hampered by the lack of small animal models of shigellosis. Here, we developed a murine model of intestinal colonization with S. sonnei. Pre-treatment of mice with antibiotics disturbed the intestinal microbiome and rendered mice susceptible to high level, gastrointestinal colonization with S. sonnei for over one week. Infection with S. sonnei CS14 harbouring a stable virulence plasmid induced an initial inflammatory response in wild type mice, with weight loss and elevated levels of fecal lipocalin 2; the S. sonnei Type III Secretion System was responsible for this inflammatory response. Expression of O-antigen and Group IV capsule by S. sonnei promoted sustained intestinal colonization, with infected mice developing mucosal and systemic antibody responses predominantly directed at these glycans. Finally, infection with S. sonnei induced a degree of protection against subsequent re-challenge. Overall, this murine model successfully mimics aspects of S. sonnei colonization and should be helpful in understanding how S. sonnei successfully survives within the gastrointestinal tract and competes with the microbiota as well as the evaluation of vaccine candidates.

Lyme arthritis, a manifestation of Lyme disease, is triggered by the spirochetal bacterium Borrelia burgdorferi (Bb), which is transmitted through the bite of the Ixodes tick. Although multiple studies have been conducted on the complex host immune response in Lyme arthritis, the spatial gene expression environment in the joint tissue remains unexplored. Here, we applied spatial transcriptomics to ankle joints of C3H mice infected with Bb, profiling tissues at peak inflammation (two weeks post infection) and after antibiotics (four weeks post-infection) during inflammation resolution. Analysis revealed spatially restricted signatures: pro-inflammatory responses dominated synovial and fibroblast populations two weeks post-infection, with elevated levels of Vimentin and I-Ek gene - and Vimentin protein - expression localized to these regions. By four weeks post-infection during the inflammation resolution phase, levels of Vimentin and I-Ek related gene and protein expression were reduced. Further, we noted an increase in the CD54+ and CD106+ double-positive population in infected mice joints compared to the vehicle treated controls. Notably, fibroblasts and synoviocytes in the medial joint regions adopted immune-like phenotypes during peak inflammation, while the same cell types in the exterior humeroradial joint displayed a more infection-resilient phenotype. These spatially resolved maps demonstrate that joint microenvironments play a crucial role in pathogenesis, offering unique insights into Lyme arthritis pathology.

A low abundance or absence of protective lactobacilli during acute trichomoniasis is a well-known phenomenon that was reported in multiple studies and is the hallmark of a T. vaginalis (TV) infection. However, a crucial question that remains unanswered is whether alterations in the lactobacilli population precede TV infection or whether the parasite plays an active role in lactobacilli disappearance. Our findings showed that TV efficiently phagocytosed the dominant Lactobacillus species L. jensenii (LJ). Phagocytosis proceeds via a pseudopodia-independent mechanism reminiscent of sinking with a preference for viable cells. The presence of viable LJ leads to an increase in secretion of 27 TV proteins, including TvGH25 lysozyme. This enzyme cleaves peptidoglycan, a major component of the bacterial cell wall. TV overexpressing TvGH25 effectively lowers the bacterial cell count, evidencing the enzyme's antimicrobial potential. These data support the notion that TV cells can suppress the Lactobacillus population through a combination of targeted secretory response and phagocytic activity, revealing novel potential targets for developing alternative therapeutic strategies against trichomoniasis.

Type III secretion systems (T3SS) are common virulence factors that facilitate the injection of anti-eukaryotic effector toxins that damage and kill target host cells. The sequence and function of the structural and regulatory proteins of these systems are conserved across Gram-negative pathogens, including Pseudomonas, Yersinia, and Salmonella. However, the identity and function of effector proteins are not conserved and are unknown in several relevant human and animal pathogens. Here, we used comparative genomics to identify and characterize the effectors encoded by Vibrio campbellii BB120, a crustacean and fish pathogen. We showed that most sequenced Vibrio strains belonging to the Harveyi clade, including V. campbellii, encode a full set of structural and regulatory genes corresponding to the T3SS1 of V. parahaemolyticus; the exception is V. natriegens. Transcriptomic and proteomic analyses identified four V. campbellii effectors that are secreted by the T3SS. Among these, two effectors are encoded outside the T3SS island, and all effector genes were co-regulated by both the master T3SS regulator ExsA and by the master quorum sensing regulator LuxR. Three effectors - VopS, CopA, and VIBHAR_06684 - exhibited toxic activity in yeast cells or bone marrow-derived macrophages, and the toxicity phenotypes were dependent on a functional T3SS. VopS and CopA are conserved among the queried species of the Harveyi clade. VIBHAR_06684 or VIBHAR_05674 did not show conservation among the queried species. These findings demonstrate that T3SSs in bacteria from the same clade have conserved structural secretion apparatuses but exhibit variance in effector repertoires. We postulate that the functions of effectors differ between species to impart roles in host specificity.

Nocardiosis is a human infectious disease caused by several species of Nocardia and primarily affecting the skin, lungs and central nervous system. The first line treatment is based on cotrimoxazole, combining trimethoprim and sulfamethoxazole. These two drugs target respectively the dihydrofolate synthase (DHFR) and the dihydropteroate synthase (DHPS) involved in the essential folate synthesis pathway. The occurrence of drug resistance to these two drugs is however frequent. While the molecular mechanisms of trimethoprim resistance are well documented in other bacteria, they remain poorly explored and documented in Nocardia. This is partly because few biochemical structural or genetic studies have been conducted on DHFR from this genus. In this study, we report the biochemical and structural characterization of DHFR from Nocardia asteroides (DHFRNad). We show that overexpression of DHFRNad in N. asteroides confers strong resistance to trimethoprim. We recombinantly expressed and purified active DHFRNad and determined its inhibition constant for trimethoprim. We solved the crystal structure of DHFRNad bound to trimethoprim at high resolution. Further, biochemical studies of mutant DHFR variants pinpointed the role of important residues for trimethoprim binding and drug-resistance. HighlightsFirst biochemical and structural characterization of Nocardia asteroides DHFR. Overexpression of DHFRNad induces high-level trimethoprim resistance in N. asteroides. Crystal structure of DHFRNad reveals key residues for trimethoprim binding. Mutagenesis confirms residues critical for trimethoprim susceptibility. IC50 data confirm strong DHFRNad inhibition by trimethoprim and methotrexate

Repressor-of-differentiation kinase 1 (RDK1) is one of two kinases expressed in bloodstream form Trypanosoma brucei parasites that were found to repress premature and spontaneous differentiation into the insect procyclic form. However, the effect of RDK1 RNAi was previously limited to the expression of a single surface coat protein, EP1 procyclin. Thus, there remains a significant gap in knowledge on the impact of RDK1 expression in bloodstream form T. brucei parasites. Here, we employ a systems biology approach and performed several proteomics analyses to identify RDK1 protein interactions and to determine the impact of loss of RDK1 expression on the bloodstream form proteome and phosphoproteome to uncover clues about potential mechanisms for RDK1 function. We found that RDK1 is dual localized to the cell membrane and the mitochondrial inner membrane with the kinase domain oriented towards the cytoplasm and mitochondrial inner membrane. Unexpectedly, the most enriched RDK1-proximal proteins were mitochondrial proteins. Furthermore, RDK1 depletion causes bloodstream form parasites to significantly upregulate many mitochondrial proteins and glycosomal proteins, several of which are upregulated in procyclic form parasites. Surprisingly, the mitochondrial phosphoproteome is largely unaffected by RDK1 depletion, while RDK1-dependent phosphoregulation is restricted to the cell membrane localization of RDK1. Lastly, we determined that RDK1 does not possess adenyl cyclase activity or alter intracellular cAMP levels; however, the dysregulated phosphoproteins correlate with functions in cyclic nucleotide signaling. In conclusion, RDK1 exhibits localization-specific kinase activity to regulate cyclic nucleotide signaling and mitochondrial proteomic maintenance in bloodstream form parasites. IMPORTANCETrypanosoma brucei is the unicellular parasite that causes African sleeping sickness and nagana disease in livestock across 36 sub-Saharan African countries. The parasite encounters different environmental niches as it is transmitted from an infected human to the tsetse fly vector as the fly takes a blood meal. T. brucei must sense environmental cues to initiate intracellular signaling pathways to promote effective differentiation and cellular remodeling from the mammalian bloodstream forms to the insect procyclic form. RDK1 is one of two kinases shown to repress premature differentiation to procyclic form, which would be detrimental for parasite survival in the human host. Therefore, it is essential to uncover mechanisms of RDK1 function to better understand how T. brucei maintains homeostasis in the human host and signals for effective cellular remodeling during parasite transmission.

Many intracellular pathogens have evolved to evade immune responses and establish a secure niche inside host cells. One such stealth pathogen is the obligate intracellular bacterium Coxiella burnetii, the causative agent of Q-fever. Coxiella translocates an array of bacterial proteins ( effectors) into the host cell through a type IVB secretion system (T4BSS) that mediates suppression of pathogen sensing and innate immunity. Yet, at a systemic level, immunocompetent hosts often restrict pathogens through Th1-mediated and cell-autonomous immunity through the expression of immune-inducible genes. However, the expression and regulation of chemokines, particularly, the CXC-ligands (CXCL9,-10,-11) that are considered biomarkers of Q-fever, is poorly understood. We observed minimal to no CXCL10 transcript levels during Coxiella infection. However, Coxiella-infected cells robustly augmented IFN{gamma}-activated expression of CXCL10 in both phagocytic and non-phagocytic cells, and this process was dependent on viability and T4BSS in epithelial cells. This phenomenon extends to other highly pro-inflammatory cytokines and other pathogens including Salmonella, Mycobacteria (H37Ra) and Toxoplasma. Synergistic increase in CXCL10 expression in Coxiella-infected, IFN{gamma}-activated cells requires ISRE and NF-{kappa}B transcriptional elements in the promoter, and the transcription factors STAT1, STAT3 and IRF9. Inhibition of STAT3 by small molecule inhibitors potently decreased the excess promoter activity of CXCL10. In addition, treatment of Coxiella-infected cells with IFN{gamma} is associated with decreased expression of SOCS1, a negative regulator of the IFN{gamma} signaling axis and relatively higher detection of extracellular bacteria. Altogether, these data demonstrate that intracellular pathogens including those conventionally considered to be "immunologically silent", robustly synergize with IFN{gamma} signaling, with STAT3 activation emerging to be a nodal point for promoting both persistent infection as well as synergism in the expression of immune genes. Author summaryAcute host immune response is often associated with production of soluble messenger molecules called cytokines/chemokines which direct the migration, recruitment and activation of leukocytes and serve as biomarkers in infectious and inflammatory diseases. The regulation of expression of these molecules and their influence on the infection process is not well-understood. In particular, interferon-gamma (IFN{gamma}), a potent pro-inflammatory cytokine produced by activated T and NK cells, activates signaling pathways involved in host defense and inflammation in macrophages and other cell types. We observed that infection with many intracellular bacterial/parasitic pathogens that employ sophisticated immune evasion strategies, synergize with IFN{gamma} signaling and significantly amplify the levels of pro-inflammatory mediators implicating the origin of adverse immune pathologies. We investigated the mechanistic basis of this seemingly counter-intuitive phenomenon, underlying host and bacterial factors involved in distinct cell types, and identified the small molecule-targetable-transcription factor STAT3 as a host determinant in promoting excess cytokine synthesis.

1.In 2024, the World Health Organisation (WHO) classified Klebsiella pneumoniae as a maximum priority pathogen for the development of new alternatives to antibiotics. In this context, understanding the regulation of key virulence mechanisms is essential. Here, we investigated the role of the orphan quorum-sensing receptor SdiA in modulating virulence-associated processes during macrophage infection. Deletion of sdiA ({Delta}sdiA) significantly increased susceptibility to phagocytosis, as demonstrated using an amoeba predation model in which mutant strains formed larger clearance zones compared to wild-type bacteria. This phenotype was also observed in murine macrophages, where {Delta}sdiA strains exhibited increased adhesion (1.5 to 2.5-fold) and phagocytic uptake. Reduced uronic acid levels were also quantified in mutant strains, indirectly indicating a diminished capsule production, likely contributing to this enhanced phagocytosis. Despite enhanced uptake, {Delta}sdiA strains showed increased intracellular survival and replication rates within macrophages, leading to reduced host cell viability. This effect occurred despite loss of interbacterial killing capacity against E. coli, suggesting that enhanced intracellular fitness is not driven by classical antibacterial offensive mechanisms. Notably, mutant-infected macrophages displayed increased generation of reactive oxygen species (ROS), NF-{kappa}B expression, and pro-inflammatory cytokines (mCXCL10 and mTNF) production, indicating that macrophage defence mechanisms are not impaired during mutant infection. Overall, bacterial survival of {Delta}sdiA could result from overwhelming, rather than actively suppressing, host defences. Together, these findings identify SdiA as a negative regulator of phagocytosis and intracellular survival in K. pneumoniae and highlight a context-dependent role in virulence. This work provides new insights into the regulatory networks governing host-pathogen interactions and bacterial adaptation to the intracellular environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/725935v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d45bfdorg.highwire.dtl.DTLVardef@e3547forg.highwire.dtl.DTLVardef@c078f9org.highwire.dtl.DTLVardef@46408a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Loss of sdiA strongly affects phagocytosis, as mutant strains showed increasing adhesion (1.5 to 2.5-fold) and phagocytic uptake. Diminished capsule production could be contributing to this enhanced phagocytosis, as reduced uronic acid levels were also quantified in mutant strains. Despite being internalized at higher rates, mutants exhibited enhanced intracellular survival and replication, reducing macrophage viability. This fitness advantage occurred independently of classical offensive mechanisms, as evidenced by a lost ability to kill E. coli. Notably, mutant-infected macrophages mounted a stronger immune response, marked by elevated ROS, NF-{kappa}B expression, and pro-inflammatory cytokines production (mCXCL10 and mTNF). Together, these findings suggest that strains survive by overwhelming, rather than suppressing, host immune defences. Created with Biorender (https://www.biorender.com/). C_FIG HighlightsO_LISdiA deletion in K. pneumoniae increases susceptibility to phagocytosis. C_LIO_LIThe mutant strains exhibit reduced uronic acid levels, indicative of capsule production. C_LIO_LISdiA mutants show enhanced intracellular survival and higher macrophage death. C_LIO_LIMutant infected macrophages have higher NF-{kappa}B, TNF, and CXCL10 responses. C_LIO_LISdiA-deficient strains lose predatory capacity against E. coli. C_LI

Coxiella burnetii is a Gram-negative, obligate intracellular pathogen and the causative agent of the zoonotic disease Q fever. Resident alveolar macrophages are the first target cells, but C. burnetii spreads to other cell types. While we have information about C. burnetii uptake and the establishment of the replication-competent phagolysosomal-like C. burnetii-containing vacuole (CCV), it is not well studied how C. burnetii exits its host cell. Here, we show that an infection with C. burnetii also triggers the activation of TFEB, a master regulator of autophagy and lysosomal development. The activation occurs in a time-dependent manner and depends on the size of the CCV. Importantly, TFEB activation during C. burnetii infection depend on MCOLN1, which channels Ca2+ across the lysosomal membrane into the cytosol. Knock-down of MCOLN1 resulted in reduced TFEB activation and smaller CCVs, while MCOLN1 activation boosted bacterial egress. Indeed, peripheral CCVs are positive for LAMP1/2 and release bacteria, without inducing host cell death. Importantly, LAMP1/2 and C. burnetii were stainable in non-permeabilized cells at sites of bacterial release, demonstrating fusion of the lysosome with the plasma membrane. Importantly, while replication of C. burnetii is not inhibited in cells lacking LAMP1/2, egress is impaired. Taken together, our data indicates that with increasing CCV size, TFEB is activated by the release of Ca2+ from lysosomes via the MCOLN1 channel, which in turn enables further CCV development and damage of the CCV membrane. This triggers lysosomal exocytosis and egress of C. burnetii without cell death induction.