Frontiers in Cellular and Infection Microbiology

Bacterial infection is a dynamic process resulting in a heterogenous population of infected and uninfected cells. These cells respond differently based on their bacterial load and duration of infection. In the case of infection of macrophages with Crohns disease (CD) associated adherent-invasive Escherichia coli (AIEC), understanding the drivers of pathogen success may allow targeting of cells where AIEC replicate to high levels. Here we show that stratifying immune cells based on their bacterial load identifies novel pathways and therapeutic targets not previously associated with AIEC when using a traditional homogeneous infected population approach. Using flow cytometry-based cell sorting we stratified cells into those with low or high intracellular pathogen loads, or those which were bystanders to infection. Immune cells transcriptomics revealed a diverse response to the varying levels of infection while pathway analysis identified novel intervention targets that were directly related to increasing intracellular AIEC numbers. Chemical inhibition of identified targets reduced AIEC intracellular replication or inhibited secretion of tumour necrosis factor alpha (TNF), a key cytokine associated with AIEC infection. Our results have identified new avenues of intervention in AIEC infection that may also be applicable to CD through the repurposing of already available inhibitors. Additionally, they highlight the applicability of immune cell stratification post-infection as an effective approach for the study of microbial pathogens.

Modern oral bacterial species present as a concoction of commensal and opportunistic pathogens originating from their evolution in humans. Due to the intricate colonization mechanisms shared amongst oral and gut bacteria, these bacteria have likely evolved together to establish and adapt in the human oro-digestive tract, resulting in the transfer of genetic information. Our liquid chromatography-with-tandem-mass-spectrometry (LC-MS-MS) analyses have revealed protein signatures, Elongation Factor Tu, RagB/SusD nutrient uptake outer membrane protein and DnaK, specifically from Porphyromonas gingivalis-containing autophagic vacuoles isolated from the infected human primary gingival epithelial cells. Interestingly, our Mass-Spectrometry analysis reported similar proteins from closely related oral bacteria, Tannerella forsythia and Prevotella intermedia. In our phylogenetic study of these key protein signatures, we have established that pathogenic oral bacteria share extensive relatedness to each other and gut resident bacteria. We show that in the virulence factors identified from gut bacteria, Elongation Factor Tu and DnaK, there are several structural similarities and conservations with proteins from oral pathogenic bacteria. There are also major similarities in the RagB/SusD proteins of oral bacteria to prominent gut bacteria. These findings not only highlight the shared virulence mechanisms amongst oral bacterial pathogens/pathobionts but also gut bacteria and elucidate their co-evolutions in the human host.

Trypanosoma cruzi is the etiological agent of Chagas disease (CD), a neglected tropical disease that affects millions of people worldwide. During its life cycle, T. cruzi undergoes several key differentiation processes that are essential for its survival. The precise mechanisms that regulate these processes remain elusive, and any interference in this cycle would represent a breakthrough in the development of effective therapy against CD. Here, after disrupting a single IP6K allele of T. cruzi, we observed that key differentiation processes (metacyclogenesis, amastigogenesis and trypomastigogenesis) were profoundly impaired. Epimastigote forms of IP6K-deficient T. cruzi exhibited morphological alterations and reduced metacyclogenesis. IP6K-deficient metacyclic forms had reduced infective potential in human cardiomyocytes. IP6K-deficient amastigote forms showed impaired ability to transform into trypomastigotes, with most of the population egressing from human cardiomyocytes without completing trypomastigogenesis. Together, our results suggest that IP6K is critical to sustain the T. cruzi life cycle. Since disruption of both IP6K alleles was lethal and the primary structure of IP6K shares only [~]15% similarity with its human homolog, this kinase emerges as a promising target for drug development against CD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/700787v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1c6e87corg.highwire.dtl.DTLVardef@1c95a1eorg.highwire.dtl.DTLVardef@3b8221org.highwire.dtl.DTLVardef@dc79fd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mycobacterium tuberculosis (Mtb) employs multiple virulence factors, including cell wall-associated proteins, to evade host immune responses. PE11, a cell wall-localized esterase, contributes to Mtb persistence by facilitating cell wall remodelling and resistance to acidic and antibiotic stress. Herein we describe a novel role of PE11 in subverting host autophagy through disruption of TFEB-mediated lysosomal function. PE11 promotes FLCN-dependent depletion of intracellular lactate to destabilize TFEB and thereby downregulate genes essential for autophagic flux and lysosomal acidification. Using a PE11-deficient Mtb strain, we demonstrate that PE11 targets the FLCN-lactate axis to regulate TFEB stability. Exogenous lactate supplementation restored TFEB stability, enhanced lysosomal acidification, and significantly reduced intracellular bacterial burden. Lactate also synergized with frontline anti-tubercular drugs to improve Mtb clearance. These findings establish PE11 as a key immune evasion factor and highlight lactate as a promising host-directed therapeutic to enhance bacterial killing and reduce antibiotic-associated toxicity.

Yersinia pseudotuberculosis is a food-borne pathogen responsible for a self-limiting gastrointestinal disease in humans known as mesenteric lymphadenitis. A phylogenetically distinct Y. pseudotuberculosis cluster from lineages 1 and 8 is associated to a specific syndrome called the Far East scarlet-like fever (FESLF), characterized by skin rash, hyperemic tongue and desquamation. Genome sequencing of FESLF strains previously revealed the presence in the plasmid pVM82 of dot/icm genes, homologous to those known to encode a T4BSS in the intracellular pathogens Legionella pneumophila and Coxiella burnetii. In the present article, we characterized the genomic features and functionality of the Y. pseudotuberculosis T4BSS (yT4BSS). We found higher dot/icm gene identity between Y. pseudotuberculosis and Pseudomonas putida genes than with those of L. pneumophila or C. burnetii. We validated the presence of all essential dot/icm genes required for the structure of a T4BSS. We then evaluated the conditions required for yT4BSS gene expression in vitro and identified an influence of temperature, with higher expression at 37{degrees}C, which mimicks the mammalian host temperature. The yT4BSS is also expressed in cellulo during the Y. pseudotuberculosis intracellular life cycle and in vivo during mouse infection. Although T4BSS functions are well characterized in the intracellular life cycle of L. pneumophila and C. burnetii, the yT4BSS appears to not be required for the intracellular survival nor for the establishment of a replication niche within cells of Y. pseudotuberculosis. Interestingly, the yT4BSS is implicated in Y. pseudotuberculosis FESLF strain pathogenicity when orally inoculated to mice but not during intravenous inoculation. Despite a role in virulence during oral infection, the yT4BSS does not influence organ colonization. However, the yT4BSS appears to be implicated in induction of important necrosis lesions in mesenteric lymph nodes and caeca of mice. Cytokine profil analyses revealed an induction of production of innate immunity related cytokines and chemokines depending on the yT4BSS in cellulo using a mouse bone marrow-derived macrophages infection model. Thus, the yT4BSS modulates cytokine responses of the host innate immune system during oral infection. In conclusion, the yT4BSS is a newly characterized virulence factor implicated in pathogenicity of Y. pseudotuberculosis strains from lineage 8 responsible for FESLF.

Bacterial vaginosis (BV) is a common infection of the lower genital tract with a vaginal microbiome dysbiosis caused by decreasing of lactobacilli. Previous studies suggested that supplementation with live Lactobacillus may benefit the recovery of BV, while the outcomes vary in people from different regions. Herein, we aim to evaluate the effectiveness of oral Chinese-origin Lactobacillus with adjuvant metronidazole (MET) on treating Chinese BV patients. In total, 67 Chinese women with BV were enrolled in this parallel controlled trial and randomly assigned to two study groups: a control group treated with MET vaginal suppositories for 7 days and a probiotic group treated with oral Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 as an adjuvant to MET for 30 days. By comparing the participants with Nugent scores [&ge;] 7 and < 7 on days 14, 30, and 90, we found that oral administration of probiotics did not improve BV cure rates (57.14% and 67.74% at day 14, 57.14% and 58.06% at day 30, 32.14% and 48.39% at day 90 for probiotic and control group respectively). However, the probiotics were effective in restoring vaginal health after cure by showing higher proportion of participants with Nugent scores < 4 in the probiotic group compared to the control group (87.50% and 71.43% on day 14, 93.75% and 88.89% on day 30, and 77.78% and 66.67% on day 90). The relative abundance of the probiotic strains was significantly increased in the gut microbiome of the probiotic group compared to the control group at day 14, but no significance was detected after 30 and 90 days. Also, the probiotics were not detected in vaginal microbiome, suggesting that L. gasseri TM13 and L. crispatus LG55 mainly acted through the gut. A higher abundance of Prevotella timonensis at baseline was significantly associated with long-term cure failure of BV and greatly contributed to the enrichment of the lipid IVA synthesis pathway, which could aggravate inflammation response. To sum up, L. gasseri TM13 and L. crispatus LG55 can restore the vaginal health of patients recovering from BV, and individualized intervention mode should be developed to improve BV cure rates.

Urinary tract infection (UTI) remains the most common infectious complication among individuals with neurogenic lower urinary tract dysfunction (NLUTD) due to spinal cord injury or disease (SCI/D). Despite widespread reliance on microbiological and symptom-based criteria for UTI diagnosis, significant ambiguity persists--especially in distinguishing clinically meaningful change from normal variability in urinary analysis results. This uncertainty contributes to overdiagnosis, inappropriate antibiotic use, and antimicrobial resistance. The present study seeks to operationalize "normal variability" of the urinary microbiome (urobiome) among adults with SCI/D. Using repeated samples collected from asymptomatic individuals over time, we analyzed inter- and intra-individual microbial composition to determine stability and fluctuation under baseline conditions. We observed wide intra- and inter-individual variability, substantial overlap between asymptomatic and pre-symptomatic states, and a consistent predominance of genera conventionally labeled as "uropathogens" even in the absence of symptoms. These findings suggest that assumptions drawn from cross-sectional studies--linking particular taxa or diversity values to health or disease--are not supported within individuals over time, at least in people with NLUTD. This study provides a foundation for distinguishing expected variation from those potentially related to infection, supporting development of precision-based diagnostic thresholds. Results offer critical insight into the ecological dynamics of the urobiome among people with NLUTD who are asymptomatic, establishes a methodological precedent for urobiome-informed clinical decision-making in SCI/D populations, and provides a foundation for distinguishing expected variation from those potentially related to infection, supporting development of precision-based diagnostic thresholds. By identifying personalized baselines and patterns of change, we aim to support research designed to obtain actionable information from the urobiome to enhance the accuracy and stewardship of UTI diagnosis and treatment in this high-risk population.

BackgroundHigh throughput 16S rRNA gene sequencing and enhanced culture methods (e.g., expanded quantitative urine culture, EQUC) have established the existence of the human bladder microbiome. We aim to test the hypothesis that the bladder environment of patients with chronic kidney disease (CKD) differs from that of unaffected controls with associated consequences for the composition of the bladder microbiome. Methods and MaterialsFemales (n=66) and males (n=66) diagnosed with CKD and age-BMI-matched healthy control (HC) females (n=22) and males (n=22) were recruited. Transurethral catheterized urine was collected for 16S rRNA gene sequencing and Expanded Quantitative Urine Culture (EQUC). Fecal samples also were sequenced. Urinary analysis, kidney function and serum cytokines were examined. ResultsBladder microbiomes of CKD females and males versus HC females and males differed (FDR<0.05); however, the difference was more obvious in females. In CKD females, sequencing revealed depletion of 5 genera, including Lactobacillus, and enrichment of 14 genera, including Escherichia/Shigella, Bifidobacterium, and several clostridial genera (FDR<0.05), while EQUC detected increased Escherichia and decreased Lactobacillus CKDB(P<0.05). Escherichia-Shigella was positively, whereas Lactobacillus was negatively, associated with CKDB-female serum creatinine (r=0.285, P=0.020; r=-0.337, P=0.006, respectively). Lactobacillus was positively associated with eGFR (r=0.251, P=0.042). Some CKD-related serum cytokines were negatively associated with clostridial genera. In contrast, the fecal microbiomes of CKD and HC females and males did not significantly differ in bacterial diversity or composition. However, bladder and fecal microbiomes of CKD females resembled each other more than those of controls, as assessed by the Bray-Curtis Dissimilarity Index (FDR<0.05). ConclusionsCKD bladder microbiomes were dysbiotic, which was more obvious in females. This dysbiosis was associated with kidney damage severity and dysregulation of serum cytokines. The increased similarity between bladder and fecal microbiomes of CKD females suggests possible "gut-leakage."

The lethal zoonosis alveolar echinococcosis is caused by the metacestode larval stage of the tapeworm Echinococcus multilocularis. During the chronic phase of the disease, metacestode tissue is growing infiltratively into liver tissue and provokes an immunes response of the host. Mechanisms of parasite defence against reactive oxygen species (ROS), which are produced during parasite growth and host immune responses, are incompletely understood so far. We herein describe the characterization of an Echinococcus Dyp (dye decolorizing) - type peroxidase, EmDyp, family members of which are typically expressed by bacteria and fungi. EmDyp showed significant homologies to bacterial and fungal Dyp peroxidases and recombinantly expressed EmDyp displayed profound enzymatic activity towards different substrates such as 3,3-diaminobenzidine or luminol. Furthermore, although structurally not being related to classical catalases, EmDyp showed catalase activity in respective activity gels. In situ hybridization experiments showed expression of the EmDyp expressing gene, emdyp, in the germinal layer of the metacestode as well as in the posterior region of the protoscolex, both in differentiated and in germinative (stem) cells of the parasite. Interestingly, RT-qPCR experiments demonstrated that emdyp expression is induced in the metacestode upon growth under aerobic conditions. Particularly high expression of emdyp was observed under in vivo growth conditions in jirds within the liver. These data indicate a role of EmDyp in the defence of the metacestode against host- and/or parasite-derived ROS during chronic alveolar echinococcosis. Since Dyp-type peroxidases are not encoded on the genomes of mammalian hosts for E. multilocularis, EmDyp might be used as a target molecule for developing novel therapeutics against the parasite.

Bacterial pneumonia is a common infection of the lower respiratory tract that can afflict patients of all ages. Multidrug-resistant strains of Acinetobacter baumannii are increasingly responsible for causing nosocomial pneumonias, thus posing an urgent threat. Alveolar macrophages play a critical role in overcoming respiratory infections caused by this pathogen. Recently, we and others have shown that new clinical isolates of A. baumannii, but not the common lab strain ATCC 19606 (19606), can persist and replicate in macrophages within spacious vacuoles that we called Acinetobacter Containing Vacuoles (ACV). In this work, we demonstrate that the modern A. baumannii clinical isolate 398, but not the lab strain 19606, can infect alveolar macrophages and produce ACVs in vivo in a murine pneumonia model. Both strains initially interact with the alveolar macrophage endocytic pathway, as indicated by EEA1 and LAMP1 markers; however, the fate of these strains diverges at a later stage. While 19606 is eliminated in an autophagy pathway, 398 replicates in ACVs and are not degraded. We show that 398 reverts the natural acidification of the phagosome by secreting large amounts of ammonia, a by-product of amino acid catabolism. We propose that this ability to survive within macrophages may be critical for the persistence of clinical A. baumannii isolates in the lung during a respiratory infection.

The role of non-caspase proteases in the regulation of programmed cell death is relatively under-studied. In this short communication, we report that infection of human gingival fibroblast (HGF) cells ex vivo by Porphyromonas gingivalis rapidly activated NF-kappa B, an antiapoptotic player; PI3 kinase was also induced, and together, they inhibited apoptosis. Later, pro-apoptotic cellular genes, including caspases, were induced. Apoptosis was evidenced by the degradation of nuclear DNA and activation of caspases. Unexpectedly, a P. gingivalis mutant that lacked all three major gingipain proteases was defective in promoting apoptosis, which was consistent with the cleavage of procaspase-3 to the active form by the culture supernatant of wild type P. gingivalis, but not that of the triple protease mutant. These results suggest that cellular death in the P. gingivalis-infected gum may be orchestrated by gingipain-regulated apoptotic factors.

The human gastrointestinal tract (GIT) is home to an abundance of diverse microorganisms, and the balance of this microbiome plays a vital role in maintaining a healthy GIT. The obstruction of the flow of bile into the duodenum, resulting in obstructive jaundice (OJ), has a major impact on the health of the affected individual. This study sought to identify changes in the duodenal microbiota in South African patients with OJ compared to those without this disorder. Mucosal biopsies were taken from the duodenum of nineteen jaundiced patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) and nineteen control participants (non-jaundiced patients) undergoing gastroscopy. DNA extracted from the samples was subjected to 16S rRNA amplicon sequencing using the Ion S5 TM sequencing platform. Diversity metrics and statistical correlation analyses with the clinical data were performed to compare duodenal microbial communities in both groups. Differences in the mean distribution of the microbial communities in the jaundiced and non-jaundiced samples were observed; however, this difference did not reach statistical significance. Of note, there was a statistically significant difference between the mean distributions of bacteria comparing jaundiced patients with cholangitis to those without. On further subset analysis, a significant difference was observed between patients with benign (Cholelithiasis) and malignant disease, namely head of pancreas (HOP) mass (p-values of 0.01). Beta diversity analyses further revealed a significant difference between patients with stone and non-stone related disease when factoring in the Campylobacter-Like Organisms (CLO) test status (p=0.048). This study demonstrated a shift in the microbiota in jaundiced patients, especially considering some underlying conditions of the upper GI tract. Future studies should aim to verify these findings in a larger cohort.

BackgroundChlamydia trachomatis is a highly prevalent sexually transmitted infection (STI) strongly associated with female infertility. Polymerase Chain Reaction (PCR) is considered the gold standard for diagnosis, utilizing primers designed to target either the ompA gene (encoding the major outer membrane protein) or the cryptic plasmid gene. MethodsThis cross-sectional study was conducted on infertile women attending the Infertility Clinic at Damascus University Obstetrics Hospital, Syria. A total of 160 cervical swab samples were analyzed from these women. Genomic DNA was extracted using a Qiagen kit, followed by PCR with primers targeting Chlamydia trachomatis major outer membrane protein gene (CTM -ompA-targeting) and cryptic plasmid (CTP -cryptic plasmid-targeting). FindingsPositivity rates were 51.3% for CTM and 31.9% for CTP. A statistically significant association was observed between C. trachomatis infection and monthly income (p= 0.039), as well as between it and education level (p= 0.001). Additionally, a significant association was found between C. trachomatis positivity and education level (p= 0.017). InterpretationThese findings underscore the significant burden of Chlamydia trachomatis in infertile Syrian women and highlight the diagnostic advantages of ompA-based PCR, suggesting socioeconomic factors influence infection prevalence.

M. tuberculosis is one of the most successful human pathogens causing tuberculosis that leads to highest daily morbidity worldwide. The evasion of the host immune responses is an important strategy that M. tuberculosis adopts. MprA (Rv0981), the response regulator of two component system is known for DNA binding activity in the pathogen and its role in persistent infection in the host. MprA is recognized as a late stage antigen during infection. A variant form of the protein MprA with G70S polymorphism (MprA*) is observed in one of our local and in several global clinical isolates of M. tuberculosis. Here we report the nuclear localization of MprA and MprA* in differentiated macrophages. MprA and MprA* increase the expression of TGF-{beta} and IL-10, the immune suppressive cytokines in THP-1 derived macrophage cells. Concurrently the phago-lysosome fusion is significantly reduced as shown by infection with M.bovis BCG. We show that single nucleotide variation in clinical isolates lead to quantitative variations resulting in host immune suppression and support the survival and persistence of the pathogen.

Toll-Like Receptor (TLR) 4, the LPS receptor, plays a central role in the control of leptospirosis and absence of TLR4 results in lethal infection in mice. Because human TLR4 does not sense the atypical leptospiral-LPS, we hypothesized that TLR4/MD-2 humanized transgenic mice (huTLR4) may be more susceptible to leptospirosis than wild-type mice, and thus may constitute a model of acute human leptospirosis. Therefore, we infected huTLR4 mice, which express human TLR4 but not murine TLR4, with a high but sublethal dose of L. interrogans serovar Copenhageni FioCruz (Leptospira) in comparison to C57BL/6J wildtype (WT) and, as a control, a congenic strain in which the tlr4 coding sequences are deleted (muTLR4Lps-del). We show that the huTLR4 gene is fully functional in the murine background. We found that dissemination of Leptospira in blood, shedding in urine, colonization of the kidney and overall kinetics of leptospirosis progression is equivalent between WT and huTLR4 C57BL/6J mice. Furthermore, inflammation of the kidney appeared to be subdued in huTLR4 compared to WT mice in that we observed less infiltrates of mononuclear lymphocytes, less innate immune markers and no relevant differences in fibrosis markers. Contrary to our hypothesis, huTLR4 mice showed less inflammation and kidney pathology, and are not more susceptible to leptospirosis than WT mice. This study is significant as it indicates that one intact TLR4 gene, be it mouse or human, is necessary to control acute leptospirosis. Contribution to the fieldDifferences of recognition exist between mouse and human TLR4, in that the anchor of LPS in the outer membrane of Leptospira (LipidA) activates murine, but not human TLR4. We hypothesized that if human TLR4 does not sense leptospiral-LPS, then transgenic mice in which murine TLR4 was replaced with human TLR4, should be more susceptible to Leptospira dissemination as compared to congenic wild-type mice, which could result in a more robust inflammatory response and pathology in the kidney. However, we found that impaired sensing of leptospiral-LPS in huTLR4 mice did not affect overall infection in comparison to wild-type mice and does not result in increased pathology of the kidney. Our study indicates that rather than leptospiral-LPS sensing, the presence of a fully functional TLR4 receptor is necessary to control acute leptospirosis.

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.

BackgroundTrypanosoma cruzi causes Chagas disease, a poorly understood and clinically heterogeneous disease. Recent work has demonstrated that parasites adapted to laboratory conditions are genomically variable, but little is known of the extent of genomic diversity from clinically isolated specimens. MethodsIn this retrospective observational genomic study, we isolated 15 T. cruzi specimens from three clinical studies of Chagas disease, representing different clinical contexts. We sequenced the genome of each strain and used single nucleotide variant (SNV) based analyses to estimate parasite genetic lineage, genomic population structure, regions of copy number plasticity, and to identify gene conversion events. In addition, we generated and annotated whole genome assemblies of each isolate. From these assemblies, we compared the repertoires of genes encoding for highly virulent and variable proteins that have been implicated in disease pathogenesis. FindingsWe identified parasites from two genetic lineages in this collection of clinical isolates. Our analysis revealed evidence of genomic instability. Diversity-generating copy number variation was statistically enriched in regions encoding the virulence-associated multigene families, while diversity-eliminating gene conversion events were enriched in regions depleted of multigene family members. We also discovered a set of multigene family members that is present in all of the clinically isolated parasite genomes and absent from all of the lab adapted strains, regardless of parasite lineage. Multigene family repertoires were more conserved among field isolated specimens of the same genetic lineage than among culture adapted strains of the same genetic type. InterpretationThis study provides whole genome sequencing data for TcV parasites isolated from naturally infected human patients with Chagas disease for the first time. Our analysis of these genomes revealed substantial genomic instability, suggesting the parasite undergoes genomic change in response to the pressures imposed by the host environment. Moreover, we observed a set of virulence-associated genes that are present exclusively within clinical isolates and absent from lab-adapted strains, indicating a potential role for these genes in parasite survival in natural hosts. These findings highlight the limitations of genetic studies focused exclusively on lab-adapted parasite strains and provide insight into the genomic features of T. cruzi that are likely to be important for clinical infection.

Trichomonas vaginalis is an extracellular parasite that inhabits the human genital tract, yet little is known about how it senses and responds to the complex vaginal microbial ecosystem. Here, we show that T. vaginalis exhibits chemotactic behavior on semisolid surfaces, forming multicellular assemblies that coordinate collective migration. Parasite colonies display both positive and negative chemotactic responses, indicating the ability to detect and react to diffusible signals. Different parasite strains display marked mutual avoidance between neighboring colonies, highlighting specific recognition mechanisms. Furthermore, we show that T. vaginalis is strongly attracted to acidic environments, revealing a niche-adapted pH taxis. Given that vaginal bacteria critically shape local pH, we examined parasite responses to representative members of the vaginal microbiota. T. vaginalis exhibited preferential chemotactic migration toward Lactobacillus gasseri, a hallmark species of eubiotic community state types (CSTs), over Gardnerella vaginalis, which is associated with dysbiotic CST-IV communities, while showing no detectable attraction to Escherichia coli. This selective migration correlated with a robust chemotactic response to lactic acid, a major metabolite produced by lactobacilli. Additionally, when the parasite is co-cultured with the equal number of L. gasseri and G. vaginalis, T. vaginalis exhibits a clear preferential binding to L. gasseri, as demonstrated by flow cytometry and fluorescent microscopy. We show that co-culture of T. vaginalis with either L. gasseri or G. vaginalis results in enhanced parasite growth only in the presence of L. gasseri. Collectively, these findings reveal pH taxis; bacteria-directed migration and preferential association with Lactobacillus as previously underappreciated behavioral traits of T. vaginalis. Such behaviors may destabilize protective microbial communities and drive the transition toward a CST-IV-type dysbiotic state which is frequently associated with trichomoniasis.

The African trypanosome, Trypanosoma brucei, disseminates systemically in tissues of the infected host resulting in complex immunopathology. The kidneys which are important in the response to the anaemia characteristic of African trypanosomiasis, are prone to acute kidney injury (AKI) from multiple noxious stimuli. Little is known about the transcriptional responses of the kidney to trypanosome infection. To assess the tissue-specific response to infection with Trypanosoma brucei, we profiled the clinicopathologic and transcriptional responses of the kidney in BALB/C (susceptible) and C57BL/6 (tolerant) murine models, at early (7 dpi) and late (21 dpi) time points of infection. Trypanosomes in the renal interstitium, tubular necrosis and inflammation characterised early infection in both mouse strains. By late infection, we observed extensive tubular necrosis in the susceptible BALB/C but reparative tubular regeneration in the tolerant C57BL/6 mice. T.b. brucei infection resulted in significant increases in serum creatinine in both strains. Consistent with the clinicopathologic findings, RNA-seq detected both mouse strain- and time-dependent transcriptional responses in the kidney. These included perturbations in genes associated with solute/ion transport, upregulation of markers of tubular injury, hypoxia, glycolysis, and a profound inflammatory and immune response, mirroring the responses observed in other models of AKI. Differential tissue pathology at late time point is preceded by expansion of CD8+ T cells, profound expression of transcription factors and upregulation of anti-inflammatory pathways in C57BL/6 mice. Our findings demonstrate that experimental T. brucei infection-induced kidney injury (TIKI) is a model of AKI and may have clinical implications for Human African Trypanosomiasis cases, who currently are not routinely screened for markers of kidney function.

Mycoplasmas are atypical bacteria that are obligate parasites of animals and plants. They are known for their capacity to contaminate and parasitize mammalian cell cultures, thereby offering fortuitous models to examine the host-microbe interaction. We used a near wildtype strain of the goat pathogen Mycoplasma mycoides (Mmc) and several minimized Mmc strains lacking genes not essential for growth in laboratory media to investigate host-mycoplasma interactions. Stains with near minimal genomes were incapable of surviving in co-culture with mammalian HEK-293T and HeLa cells. We identified a cluster of eight non-essential genes that when added back to the minimized strains enabled mycoplasma attachment to mammalian cells. Those genes did not restore the capacity of the minimal cell to grow when co-cultured with mammalian cells indicating processes of attaching to mammalian cells and parasitism involve different genes. Furthermore, minimized Mmc strains were not phagocytized by human myeloid cells unlike the near wildtype Mmc.