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Bordetella pertussis is a strictly human, re-emerging respiratory pathogen and the causative agent of whooping cough. Through its adaptation to humans, B. pertussis decayed or lost genes of the sulfate assimilation pathway and, consequently, must obtain cysteine from the host. Previously, we showed that the sulfur metabolism of B. pertussis is substantially rewired during infection of human macrophages. Here, we investigated the role of several cysteine metabolism- and transport-related genes in the fitness and virulence of this pathogen. We show that excess cysteine strongly induces the expression of genes encoding two cysteine dioxygenases, BP2871 and BP3011, and a putative sulfite exporter, BP2808. The mutant lacking both cysteine dioxygenase genes exhibits impaired growth in vitro, severely reduced secretion of pertussis toxin, and attenuated virulence in vivo. We also demonstrate the essential role of the sulfite exporter BP2808 and {gamma}-glutamyl-cysteine synthase BP0598 in the adaptation of B. pertussis to stress induced by excess cysteine. Intriguingly, both cysteine dioxygenases contain cysteine near the active site, a feature typical of mammalian enzymes and associated with the capacity to increase CDO activity and stability in response to excess cysteine. We hypothesize that the presence of cysteine represents an evolutionary adaptation that improves the survival of B. pertussis within a mammalian host. Overall, our data suggest that sulfur metabolism has been effectively streamlined in B. pertussis and plays an important role at the host-pathogen interface. ImportanceSulfur is one of the essential nutrients required by cells for growth and cysteine is central to sulfur metabolism. While most bacteria prefer environmentally available sulfate as their cysteine source, several bacterial pathogens rely on cysteine provided by the host. Here we show that Bordetella pertussis, the causative agent of whooping cough, has simplified its sulfur metabolism. Our data suggest that two cysteine dioxygenases and sulfite exporter play key roles in sulfur homeostasis and redox balance. Both dioxygenases enable the pathogen to use cysteine as a source of sulfur and the sulfite exporter removes the toxic byproduct of cysteine conversion. Importantly, lack of cysteine dioxygenase activity leads to aberrant secretion of pertussis toxin, one of the essential virulence factors, resulting in attenuated virulence of the pathogen. We suggest that cysteine auxotrophy can be considered part of an infection strategy that assists B. pertussis in adapting to its human host.

Autophagy is a critical cellular process regulated by Atg proteins, yet its modulation by redox-active compounds and iron remains incompletely understood. Here, we investigated the effects of dithiothreitol (DTT) and iron on autophagy and on AaAtg4 protease activity in the plant-pathogenic fungus Alternaria alternata. Using GFP-tagged AaAtg8, fluorescence microscopy and proteolysis assays revealed that DTT markedly enhanced autophagic vacuole formation and GFP release, indicating increased autophagic flux. Western blot analyses confirmed that DTT promoted AaAtg8 lipidation, while co-treatment with hydrogen peroxide (H2O2) suppressed this modification. AaAtg4 was constitutively active and could process AaAtg8 regardless of DTT supplementation, whereas moderate DTT concentrations elevated AaAtg4 protein abundance and phosphorylation. Bimolecular fluorescence complementation assays demonstrated that DTT, but not iron, facilitated AaAtg4-AaAtg8 interactions and vacuolar localization, whereas H2O2 counteracted these effects. Notably, combined DTT and H2O2 sustained autophagy at a low but stable level, suggesting a redox balance in autophagic regulation. Iron supplementation selectively destabilized AaAtg8 and modulated AaAtg4 phosphorylation in a concentration-dependent manner, without altering autophagy or protease activity. Collectively, these findings demonstrate that DTT enhances autophagy primarily by promoting AaAtg8 lipidation, AaAtg4 phosphorylation, and AaAtg4-AaAtg8 complex formation, while exerting minimal influence on AaAtg4 protease activity. In contrast, ion regulates autophagy flux through its effects on AaAtg4 phosphorylation and AaAtg8 stability, without significantly altering AaAtg4 protease activity, AaAtg8 lipidation, or AaAtg4-AaAtg8 interactions. Together, this work underscores the intricate interplay between redox signaling, nutrient cues, and autophagy regulation in A. alternata. IMPORTANCEThis study provides critical new insights into how redox-active compounds and iron modulate autophagy in the plant-pathogenic fungus Alternaria alternata, a pathogen of agricultural relevance. By dissecting the distinct roles of DTT, hydrogen peroxide, and iron in regulating AaAtg8 lipidation, AaAtg4 phosphorylation, and AaAtg4-AaAtg8 interactions, our findings reveal that autophagy is not simply a constitutive process but is finely tuned by redox balance and nutrient cues. This work advances the fundamental understanding of autophagy regulation in filamentous fungi, highlights the interplay between oxidative stress and protease activity, and establishes a framework for exploring how environmental factors shape fungal pathogenicity. Ultimately, these insights may inform novel strategies to mitigate crop fungal diseases by targeting autophagic pathways.

As Earths temperature rises, fungal pathogens are adapting, altering host-pathogen interactions, disease patterns, and response to the antimicrobial drugs. Here, we show that thermal adaptation to 42{degrees}C leads to reversible changes in fungal colony size, appearance, and azole drug response in the human pathogenic fungus Aspergillus fumigatus. Importantly, this adaptation is mediated by a lncRNA, afu-182, whose RNA levels negatively correlate with temperature. Growth at a lower temperature or ectopic upregulation of afu-182 RNA levels reverses the temperature adaptation. Global transcriptomic analyses show an enrichment of pathogenesis-associated genes at 37{degrees}C and 42{degrees}C compared to 25{degrees}C. Interestingly, we found that small heat shock proteins and chaperones, but not ATP-dependent heat-shock proteins, are negatively regulated by afu-182 at 37{degrees}C and 42{degrees}C at transcriptional level. Previously, we have shown that {Delta}afu-182 strains produce worse disease outcomes in a murine model of invasive pulmonary aspergillosis (IPA). Here, more importantly, we show that the overexpression of afu-182 in clinically azole-resistant isolates increased survival in a murine model of IPA. Taken together, fungal adaptation to increased temperature leads to a decrease in afu-182 RNA levels that is associated with worse disease outcomes upon azole treatment. This provides a framework to take temperature into account when analyzing the rise in azole MIC in environmental and clinical isolates. Significance statementAspergillus fumigatus is the causative agent of most mold associated infections and can tolerate temperatures above 50{degrees}C. A lncRNA levels negatively correlate with increasing temperature, and this increases the fungis ability to tolerate azole drugs both in vitro and in vivo. Changing the levels of afu-182 improves anti-fungal treatment outcomes.

The envelope of Gram-negative bacteria like Escherichia coli is multilayered with two membranes sandwiching a peptidoglycan cell wall. The inner membrane is a typical phospholipid bilayer whereas the outer membrane is asymmetric with phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. We recently discovered that inactivation of the conserved peptidoglycan synthesis machinery responsible for cell elongation causes defects in both peptidoglycan and LPS synthesis in E. coli. This finding suggests that the isolation of suppressors that rescue the growth phenotype caused by an impaired cell elongation system is an attractive means of identifying factors involved in coordinating the biogenesis of different envelope layers. Here, we report the results of a global, transposon sequencing-based screen for such suppressors. The inactivation of a number of factors including the phospholipid synthesis enzyme PlsX was found to partially suppress the growth defects of a cell elongation mutant. Deletion of plsX also conferred increased resistance to CHIR-090, an inhibitor of the committed step of LPS synthesis catalyzed by LpxC, suggesting that loss of PlsX function stimulates LPS synthesis. Evidence is presented that increased CHIR-090 resistance is not mediated by changes in the activity of the proteolytic system (YejM-LapB-FtsH) controlling LpxC turnover. Rather, our results are consistent with a model in which the phospholipid precursor acyl-phosphate produced by PlsX serves as an inhibitor of LpxC to lower the rate of LPS synthesis when phospholipid synthesis capacity is reduced. IMPORTANCEOver the last several decades, most proteins essential for Gram-negative cell surface assembly have been characterized. However, relatively little is known about how the synthesis of different envelope layers is coordinated to promote uniform surface growth. Here, we report the results of a transposon sequencing-based genetic screen for mutants that suppress defects in the conserved peptidoglycan synthesis machinery responsible for cell elongation. Inactivation of the plsX gene encoding a phospholipid synthesis enzyme was found to both suppress the growth defect of a cell elongation mutant and to confer elevated resistance to an inhibitor of lipopolysaccharide synthesis. Our results suggest the attractive possibility that the product of PlsX, acyl-phosphate, may play a regulatory role in coordinating the phospholipid and lipopolysaccharide synthesis pathways.

Staphylococcus aureus is one of the most frequently co-isolated pathogens in polymicrobial infections, where interspecies interactions contribute to enhanced virulence, persistence, and antimicrobial tolerance. Nutrient availability plays a central role in these interactions as microorganisms compete for resources required to sustain essential cellular processes. For instance, branched-chain amino acids (BCAAs) are critical for protein synthesis, and valine synthesis pathway precursors are essential for energy production. In S. aureus, BCAAs are also the precursors for branched-chain fatty acids (BCFAs), the dominant fatty acids in the S. aureus membrane. We previously identified a second pathway that uses branched-chain carboxylic acids (BCCAs) and the high-affinity acyl-CoA synthetase MbcS to catalyze the synthesis of BCFA precursors. However, the physiological role of this pathway and the conditions triggering its activation remain unclear. Here, we show that mbcS is restricted to S. aureus and closely related human-associated staphylococci. Phylogenetic analyses suggest that MbcS arose from a refunctionalization event and represents a non-orthologous replacement for the phosphotransbutyrylase (Ptb) and butyrate kinase (Buk) enzymes. Consistent with this model, Ptb and Buk from Staphylococcus pseudintermedius catalyze the formation of branched-chain acyl-CoAs from BCCAs, but only at high substrate concentrations. We further show that mbcS expression is upregulated in a codY mutant, implicating this pathway in BCAA-limited conditions. In support, we show that mbcS is required for optimal fitness during intra-species competition. Together, our findings support a model in which the MbcS-dependent pathway enables S. aureus to scavenge BCFA precursors under nutrient-limited conditions, providing a competitive advantage in polymicrobial environments. ImportanceStaphylococcus aureus is a major contributor to polymicrobial infections, where competition for nutrients can influence bacterial physiology and survival. A deeper understanding of how S. aureus adapts to nutrient limitation is therefore essential to explain its success as a human pathogen. In S. aureus, the acyl-CoA synthetase MbcS supports BCFA synthesis from BCAA-derived carboxylic acids and aldehydes, which are released into the environment as by-products of bacterial metabolism. Herein, we provide evidence that S. aureus acquired the acyl-CoA synthetase MbcS as an adaptive trait. This metabolic innovation allows this bacterium to maintain membrane homeostasis under nutrient limitation and compete against neighboring bacteria. Our findings highlight an adaptive strategy that may contribute to the persistence of S. aureus in polymicrobial infections.

The cell wall of the Gram-positive bacterium Enterococcus faecalis is decorated with the enterococcal polysaccharide antigen (EPA), consisting of a core rhamnan backbone linked covalently with a strain-variable teichoic acid-like (TA) polymer. Current models propose that the TA decoration is a repeating polymer composed of two alternating subunits, designated TAI and TAII, which are attached to the rhamnan core via a mild-acid labile phosphodiester bond from the initiating TAI subunit. In this study, we characterize the EpaU autolysin encoded within the EPA biosynthetic gene cluster. We demonstrate that the cell wall-binding domain of EpaU associates with the intact TA domains of EPA synthesized with the aid of the glycosyltransferases EpaR and EpaX. We further show that EpaU is a potent autolysin that binds generally over the E. faecalis cell surface, suggesting that it functions as a remodeling peptidoglycan hydrolase. The absence of EpaU leads to increased ampicillin resistance and elevated intracellular levels of the second messenger c-di-AMP. These data suggest that E. faecalis possesses a mechanism that senses the integrity of the peptidoglycan meshwork and employs c-di-AMP to regulate cell turgor, potentially altering the antibiotic resistance. ImportanceEnterococcus faecalis is an important opportunistic pathogen that can cause severe nosocomial infections. Knowledge of how bacteria remodel the cell wall is key to understanding many important cellular processes, such as antibiotic resistance, cell division, biofilm formation, and stress resistance. In this study, we shed new light on the structural details of the main cell wall polysaccharide, Enterococcal Polysaccharide Antigen (EPA), and its interaction with EpaU, an autolysin that cleaves peptidoglycan during cell wall remodeling. We also report a link between EpaU and the regulation of turgor pressure via cyclic dinucleotide signaling. This work contributes to a more complete picture of E. faecalis cell wall and may provide insight into the development of antimicrobial agents based on autolysins.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes pulmonary infection in immunocompromised patients, which in severe cases leads to fatal meningoencephalitis. Cryptococcus exhibits unique glycobiology that plays important roles in pathogenesis. Unlike model yeast and other common fungal pathogens, Cryptococcus incorporates xylose, a five-carbon monosaccharide, into its glycans. One trimer motif, which consists of xylose in {beta}-1,2 linkage to the reducing mannose of an -1,3-mannose dimer, occurs in key cryptococcal glycoconjugates that include protein N- and O-linked glycans, glycosylinositol phosphorylceramides (GIPCs), and the capsule polysaccharides glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal). We previously identified cryptococcal {beta}-1,2-xylosyltransferase 1 (Cxt1), which catalyzes formation of this motif in GIPCs, GXM, and GXMGal. Here, we report the discovery of a second enzyme, cryptococcal {beta}-1,2-xylosyltransferase 2 (Cxt2). Through characterization of cells that lack one or both corresponding genes (CXT1 and CXT2), we have dissected the biological roles of these enzymes, which are overlapping but not identical. Notably, Cxt1 and Cxt2 co-localize in the Golgi, influence capsule in a strain-dependent manner, and together are responsible for all xylose addition to O-glycans. Overall, our work highlights unique roles of these two enzymes and fills a gap in understanding of cryptococcal glycan synthesis. IMPORTANCECryptococcus neoformans is an opportunistic fungal pathogen that causes almost 150,000 deaths each year worldwide. Cryptococcus synthesizes unique glycan structures that play important roles in its biology and pathogenesis. One abundant component of these structures is xylose, a five-carbon monosaccharide. Because xylose is not used by many fungal organisms, including model yeast, we have limited information about how cells add it to their glycans. Here we report a xylosyltransferase enzyme that performs this function, and we characterize specific biological roles of this protein and a closely related one we discovered earlier. We find that these proteins together perform all detectable xylose addition to an important class of protein-linked glycans (O-glycans). They also both participate in other synthetic processes, although this varies with the specific enzyme and strain background. These results contribute to our understanding of cryptococcal glycan synthesis and underscore the importance of testing multiple background strains.

Living organisms must adequately respond to stress to survive and proliferate. Bacterial pathogens face multiple stressors during infections, including oxidative stress from host innate immune cells and antibiotic treatment from clinical therapy. The pathogenic bacterium Acinetobacter baumannii is considered the most critical threat to public health due to its broad antibiotic resistance. However, it is poorly known how A. baumannii properly responds to antibiotics and stress molecules during infection. Here, we investigate the mechanisms by which A. baumannii regulates its morphology to reduce the uptake of stress molecules under oxidative stress and antibiotic exposure, thereby conferring virulence and survival during infection. The transcriptional regulator IscR responds to oxidative stress by upregulating pbp1a, which encodes an enzyme involved in peptidoglycan biosynthesis. Under oxidative stress, bacteria undergo a morphological shift from a rod to a coccoid form, reducing their surface area and thus decreasing their absorption of reactive oxygen species. Inactivation of either iscR or pbp1a results in an elongated morphology characterized by an elevated surface area, thereby reducing A. baumannii survival under oxidative stress. Furthermore, IscR-mediated morphological control is essential for survival under antibiotic treatment. Moreover, IscR-mediated morphology regulation is required for A. baumannii survival in macrophage and mouse models. These findings elucidate a strategy by which A. baumannii uses IscR to adapt to stress through morphological control, facilitating its survival during infections against both immune response and antibiotic therapy. IMPORTNACEAcinetobacter baumannii is a major cause of nosocomial infections. It poses a critical threat due to its extensive antibiotic resistance. This study reveals that the pathogen can change its cellular shape to survive immune system attacks and antibiotic treatment. This change represents a previously unknown survival strategy. A. baumannii transitions to a coccoid morphology under oxidative stress and antibiotic treatment. It does so by activating the peptidoglycan synthesis gene pbp1a through the IscR transcriptional regulator. This rapid morphological adaptation helps A. baumannii evade host defenses and resist antibiotic treatment by reducing uptake of stress molecules. Our findings advance understanding of how pathogens adapt to hostile environments and identify new therapeutic targets. By blocking this shape remodeling ability, it may be possible to render pathogenic bacteria more vulnerable to immune responses and antimicrobial treatments. This offers a promising strategy for combating this multidrug-resistant pathogen.

The saprophytic mold Aspergillus fumigatus produces small (2-3 {micro}m) airborne spores (conidia) that can reach the lung alveoli upon inhalation. There, they encounter the surfactant-rich environment of the alveolar epithelium and initiate swelling and germination. Primary alveolar macrophages are essential for the rapid clearance of conidia and maintenance of pulmonary homeostasis. However, A. fumigatus remains the leading cause of invasive pulmonary aspergillosis in immunocompromised patients, and the mechanisms governing fungal clearance versus invasion remain poorly understood. In this study, we adapted a previously established monocyte-derived alveolar-like macrophage (ALM) model to investigate early host-pathogen interactions upon A. fumigatus challenge. Given the requirement of GM-CSF for maintaining alveolar macrophage identity and function, we included GM-CSF differentiated macrophages (GM-M), as a widely used reference model. Primary alveolar macrophages (pAM), isolated from human lung biopsies were used to validate the physiological relevance of the ALM model. Combined phenotypic, functional and transcriptomic analyses demonstrated that ALMs closely resemble pAMs under both steady-state and infection conditions across multiple time points and fungal burdens. Notably, fungal dual RNA-sequencing revealed a significant upregulation of fungal virulence-associated factors during interaction with ALM, which was not observed in GM-M co-cultures. Collectively, these findings support the use of ALMs as a robust, experimentally accessible and physiologically relevant in vitro model for investigating early A. fumigatus infection, providing new insights into host-pathogen dynamics at the alveolar interface.

Francisella tularensis is a highly infectious, Gram-negative intracellular bacterium and the causative agent of tularemia, a potentially fatal disease. Owing to its low infectious dose, ease of aerosolization, high virulence, lack of an effective vaccine, and potential use as a bioterrorism agent, F. tularensis is classified by the CDC as a Tier 1 Category A Select Agent. Despite its clinical importance, the mechanisms underlying F. tularensis virulence remain incompletely understood. In this study, we generated a partial Tn5 transposon insertion mutant library in the F. tularensis live vaccine strain (LVS) and identified a mutant disrupted in the FTL_0690 gene through screening under macrophage-like conditions. FTL_0690 encodes an acyl-CoA synthetase. Characterization of both a transposon-insertion mutant and a targeted deletion mutant ({Delta}FTL_0690) revealed critical roles for this enzyme in F. tularensis pathobiology. Loss of FTL_0690 increased sensitivity to oxidative stress and impaired intracellular growth within macrophages compared to wild-type F. tularensis LVS. Lipidomic profiling of the {Delta}FTL_0690 mutant revealed disruptions in fatty acid metabolism, membrane lipid remodeling, and redox homeostasis. Altered lipid-derived and membrane-associated metabolites indicated defective phospholipid incorporation and altered membrane composition, likely contributing to oxidative stress sensitivity and reduced intramacrophage survival. Collectively, these findings demonstrate that FTL_0690 which encodes long-chain acyl-CoA synthetase, contributes to lipid homeostasis, membrane integrity, and oxidative stress resistance of F. tularensis. ImportanceThis work addresses critical gaps in our understanding of Francisella tularensis virulence by identifying lipid metabolism as a central determinant of intracellular survival and stress resistance. By integrating transposon mutagenesis, targeted gene deletion, and lipidomic profiling, this study provides mechanistic insight into how metabolic remodeling supports pathogenesis. Our identification and characterization of FTL_0690 as a long-chain acyl-CoA synthetase essential for lipid homeostasis, membrane integrity, and oxidative stress resistance reveals a previously unappreciated link between fatty acid metabolism and intramacrophage survival of F. tularensis.

While pathogenic fungi can acquire resistance to the current arsenal of antifungals through genetic mutations, heteroresistance has emerged as an important new cause of therapeutic failures. Heteroresistance is generally thought to arise in small subpopulations that display phenotypic resistance to antifungals without genetic mutations. This study blurs that line by showing gain-of-resistance mutations in FKS2, which encodes a target of echinocandins and fungerps, cause large amounts of heteroresistance in Candida glabrata through heterogeneous expression of the gene even in clonal cell populations. Heteroresistance decreased when stress-responsive transcription factors (Crz1, Rlm1) were eliminated and was nearly abolished when the upstream regulators (calcineurin, Slt2) were mutated or inhibited. Identical gain-of-resistance mutations in FKS1, a paralog of FKS2, showed much less heteroresistance due to its constitutive expression coupled with variable levels of antagonism by wild-type FKS2. A genome-wide screen using Tn-seq revealed additional regulators of heteroresistance and resistance including IRA1, an inhibitor of the Ras1-PKA signaling pathway that senses glucose availability. IRA1 increased expression of FKS2 and decreased expression of FKS1, which increased heteroresistance and decreased resistance, respectively, when these genes carried resistance mutations. Similar principles may govern heteroresistance in other fungal pathogens such as Candida parapsilosis, which naturally carries resistance mutations in FKS1 and frequently exhibits heteroresistance to echinocandins, and Candidozyma auris, which easily acquires such mutations.

Urinary tract infections (UTIs) are a significant public health burden that impact millions of people every year and are highly prevalent among in hospital-acquired infections. Klebsiella pneumoniae is the second most common cause of UTIs after uropathogenic Escherichia coli (UPEC). Thus far, the molecular mechanisms underlying pathogenesis is better understood in UPEC than K. pneumoniae. UPEC is known to have fitness factors such as fimbrial adhesion and evasion of complement-mediated killing. In other infection types, K. pneumoniae fitness has been associated with mucoidy and diverse capsular serotypes. To establish K. pneumoniae virulence factors contributing to UTI, we examined how environmental cues regulate urovirulence-associated phenotypes in clinical K. pneumoniae UTI strains. These factors included capsular polysaccharide properties, hemagglutination, serum resistance, adherence to bladder epithelial cells, and in vivo fitness. We found that clinical K. pneumoniae UTI isolates phenotypes are highly heterogeneous and can change in response to human urine. Despite K. pneumoniae clinical isolates presenting heterogeneous fitness properties, all similarly colonize the urinary tract. These results suggest that additional fitness factors contribute to K. pneumoniae uropathogenesis. Identifying these shared fitness factors will provide mechanistic insights into Klebsiella uropathogenesis and reveal candidate therapeutic targets.

Lipoteichoic acid is an essential cell envelope polymer in Staphylococcus aureus and contributes to beta-lactam resistance in methicillin-resistant S. aureus. Mutations that alter lipoteichoic acid synthesis sensitize MRSA to beta-lactam antibiotics, but the mechanism connecting envelope polymer defects to antibiotic susceptibility has remained unclear. Here, we used suppressor genetics, genome sequencing, transcriptomics, inducible gene expression, antibiotic susceptibility assays, and measurements of intracellular potassium, cyclic di-AMP, and penicillin-binding protein production to define this connection. We found that lipoteichoic acid synthesis defects increased expression of a ParB-like protein and the mechanosensitive channel MscS. Repression of this locus in RNA polymerase suppressor mutants restored beta-lactam resistance, whereas induced expression of both genes reduced resistance. Increased ParBL-MscS expression was associated with decreased intracellular potassium and reduced cyclic di-AMP, linking altered membrane-envelope physiology to second messenger signaling. Lipoteichoic acid synthesis mutants also showed reduced production of penicillin-binding protein 4, an important determinant of beta-lactam resistance. Modest restoration of penicillin-binding protein 4 improved resistance in the mutant with reduced lipoteichoic acid abundance, whereas the mutant producing elongated lipoteichoic acid showed a distinct response, indicating that different lipoteichoic acid defects impose different envelope stresses. Together, these findings identify a potassium-dependent pathway connecting lipoteichoic acid synthesis, mechanosensitive channel activity, cyclic di-AMP signaling, penicillin-binding protein 4 production, and beta-lactam resistance. This work reveals how cell envelope perturbations are converted into cytoplasmic regulatory responses that control antibiotic susceptibility and suggests that ion homeostasis and cyclic di-AMP signaling may be exploitable pathways for restoring beta-lactam efficacy against MRSA. Author SummaryAntibiotic-resistant Staphylococcus aureus, including methicillin-resistant S. aureus, is difficult to treat because it can withstand many beta-lactam antibiotics, a widely used class of drugs. Previous work showed that changes in lipoteichoic acid, an important molecule in the bacterial cell envelope, make these bacteria more sensitive to beta-lactams. However, it was not clear how a change at the cell surface affects antibiotic resistance inside the cell. In this study, we found that defects in lipoteichoic acid production activate a pathway involving a predicted membrane channel. This pathway changes the level of potassium inside the bacterial cell and affects a small signaling molecule that helps coordinate cell wall maintenance. These changes also reduce the amount of an enzyme involved in building the cell wall, making the bacteria more vulnerable to beta-lactam antibiotics. Our findings suggest that antibiotic resistance depends not only on the direct targets of antibiotics, but also on how bacteria maintain the balance between their cell envelope, ion levels, and internal signaling. Understanding this connection may help identify new ways to make resistant bacteria more sensitive to existing antibiotics.

Toxoplasma gondii is a single-celled parasite belonging to the Apicomplexa phylum. Toxoplasmas single mitochondrion is highly dynamic, changing its morphology as the parasite undergoes egress and invasion. Recently, we have demonstrated that mitochondrial morphology is driven by a protein named Lasso Maintenance Factor 1 (LMF1). This protein interacts with IMC10, a protein present at the parasites inner membrane complex (IMC), mediating a unique membrane contact site between the IMC and mitochondrion. Interestingly, parasites lacking either LMF1 or IMC10 have abnormal mitochondrial morphology, cell division defects, and delayed propagation in tissue culture. Although both components of the tether were identified, the functions of this contact site remain unknown. In this work, we show that {Delta}lmf1 parasites exhibit upregulation of egress signaling and downregulation in folate metabolism and pantothenate biosynthesis. {Delta}lmf1 parasites exhibit increased intracellular calcium levels, leading to greater sensitivity to ionophore-induced egress and microneme secretion. We have confirmed that parasites have decreased levels of tetrahydrofolate and coenzyme A, showing a limitation in cofactor production. Interestingly, the {Delta}lmf1 parasites prefer glutamine instead of glucose as a catabolic substrate. Accordingly, we demonstrate for the first time that proper mitochondrial positioning is crucial for folate and Coenzyme A metabolism as well as egress signaling. IMPORTANCEToxoplasma gondii is the causative agent of Toxoplasmosis, a disease that affects a third of the worlds population. This parasite has a single, highly dynamic mitochondrion. The parasites mitochondrion changes shape depending on environmental conditions (inside or outside the host cell) or on stressors, such as drugs. Our laboratory characterized the proteins involved in regulating mitochondrial dynamics in the parasite, but the functional importance of these mitochondrial changes has not yet been described. Here, we show that the shape of Toxoplasmas mitochondrion is important for the synthesis of key cofactors, such as folates and coenzyme A. We show that mitochondrial shape in this parasite is important for signaling the parasites exit from the host cell, a critical process in its life cycle. These findings review a previously unknown function of a parasite-specific organelle contact site, providing new insights into the importance of mitochondria for these parasites.

Cerebral malaria is a major complication of Plasmodium falciparum infection that occurs upon the sequestration of infected red blood cells (iRBCs) in brain capillaries, resulting in the loss of endothelial barrier integrity, brain swelling, and frequently long-term sequelae or death. P. falciparum-iRBCs cause the disruption of human brain microvascular endothelial cell barrier integrity in vitro, mimicking the microenvironment of cerebral malaria, yet the specific mechanisms mediating this process remain unknown. Upon infection of the host RBCs, P. falciparum produces hemozoin, a crystal form of heme generated following the degradation of hemoglobin by the parasite. Here we show that the endothelial barrier-disrupting activity is found entirely in the hemozoin fraction of P. falciparum-iRBCs. This activity is not caused by the hemozoin crystal itself, which is not able to induce barrier disruption, but by the biomolecules that are associated with it. Treatment of purified P. falciparum hemozoin with proteases inhibits the disruption of endothelial barrier integrity caused by the hemozoin, indicating an important role for proteins in the disruption of the barrier. Conversely, treatment with nucleases did not affect hemozoin barrier disrupting activity. These results identify a key molecular mechanism in the P. falciparum-mediated brain endothelial barrier disruption during cerebral malaria and may open new avenues for the treatment of this complication. IMPORTANCEWhile several specific biomolecules have been proposed to contribute to the disruption of endothelial barrier integrity in cerebral malaria, no single P. falciparum- or host-derived factor has been definitively identified as the primary driver of this disruption. Here, we identify the brain endothelial barrier-disruptive P. falciparum-iRBC-derived activity to be caused by biomolecules bound to hemozoin, identifying a key, novel mechanism in the pathogenesis of cerebral malaria. The finding that P. falciparum hemozoin also disrupts a pulmonary endothelial cell barrier opens the possibility that this mechanism underlies other severe malaria complications. The implication of P. falciparum-iRBC-derived proteins in this mechanism is in line with previous reports, providing a novel interpretation of these findings in the context of hemozoin-binding. This knowledge provides a new perspective in the search for specific molecules and mechanisms involved in barrier disruption, which may lead to the development of much-needed specific treatments for cerebral malaria.

To establish infection, uropathogens must overcome several host defenses including the glycosaminoglycan (GAG) layer coating the apical surface of the bladder urothelium. GAGs are thought to protect against urinary tract infection (UTI) by serving as scaffolding sites for commensals, providing barrier function and preventing uropathogen adherence. However, the ability of uropathogens to degrade and utilize GAGs and the contribution of these activities toward UTI progression is largely unknown. We previously discovered that the uropathogen Proteus mirabilis, a common cause of catheter-associated UTI (CAUTI), degrades the GAG chondroitin sulfate (CS). In this study we sought to define the kinetics and regulation of CS degradation by diverse P. mirabilis strains clinically isolated from both recurrent UTI and CAUTI patients. We found variation in CS degradation kinetics between P. mirabilis strains and media types. However, CS degradation depended on conserved putative chondroitin sulfate ABC endo- and exolyases in all strains. Furthermore, we found that CS degradation in Pm123 was repressed by urea and that this repression was dependent on P. mirabilis urease activity. Complementation of the Pm123 endolyase into urea-insensitive HI4320 resulted in a urea-sensitive CS degradation phenotype suggesting functional differences between the Pm123 and HI4320 endolyases. Sequence alignment and structural modeling analysis identified two unique point mutations within the Pm123 endolyase that may contribute to urea sensitivity. Finally, unlike urea-insensitive P. mirabilis strains, Pm123 demonstrated attenuated swarming and loss of chondroitin endolyase activity had no effect on Pm123 virulence in a mouse CAUTI model. Our results suggest that the kinetics and regulation of CS degradation differ between P. mirabilis strains and in urea-sensitive strains, thus reduces the contribution of CS degradation to urovirulence during murine CAUTI. ImportanceThis work demonstrates that the ability to degrade a common component of bladder mucosal surfaces, chondroitin sulfate, is a phenotype that is shared by multiple strains of the common catheter-associated UTI (CAUTI) pathogen P. mirabilis. We find that this activity is dependent on encoded chondroitin ABC endo- and exolyases, first described in Proteus vulgaris. Additionally, we discovered that for P. mirabilis strain Pm123, degradation of CS is negatively regulated by the presence of urea, a major component of urine. The repression of CS degradation by urea is dependent on the activity of the P. mirabilis urease enzyme, which breaks down urea producing ammonia which raises pH. We found expression of the Pm123 CS endolyase was sufficient to confer a urea-sensitive CS-degradation phenotype and identified two unique mutations within the Pm123 enzyme that may contribute to urea sensitivity. Finally, we find that while CS-degradation plays a role in progression and severity of murine CAUTI model in urea-insensitive P. mirabilis, there was not significant difference in CAUTI outcomes between the urea-sensitive Pm123 wild-type and chondroitinase knockout strains. This study represents a major step forward in understanding the diversity of CS degradation activity and regulation among clinical strains of the critically important CAUTI pathogen P. mirabilis as well as its contribution to urovirulence.

Salmonella enterica encounters acid stress during gastrointestinal transit and within the phagosomal environment of macrophages. Acid stress resistance has been well characterized in Salmonella enterica serovar Typhimurium, but comparative studies in the human-adapted Salmonella enterica serovar Typhi are limited. We compared the growth of S. Typhimurium 14028s and S. Typhi Ty2 at pH values ranging from 3-8 and observed that Salmonella enterica serovar Typhimurium exhibits enhanced growth at pH 4.5 compared to S. Typhi. Comparative transcriptomic profiling of S. Typhimurium and S. Typhi at pH 4.5 and 7.5 identified numerous differentially expressed acid-induced genes (DEGs), including genes encoding membrane proteins (OmpC, PhoE, HydB), a transcriptional regulator (RpoS), and stress response proteins (YciG, STM14_1829, YmdF). Targeted deletion of selected genes in S. Typhimurium significantly suppressed growth at acidic pH, confirming their role in acid stress resistance. These resistance mechanisms are compromised in S. Typhi due to pseudogenization. Heterologous expression of pseudogenized genes in S. Typhi restored acid tolerance. Collectively, these findings suggest that S. Typhi has lost the ability to withstand acid stress due to genomic decay and the loss of multiple genes essential for acid survival in S. Typhimurium, reflecting divergent evolutionary paths in these two serovars. ImportanceSalmonella Typhimurium must adapt to acidic pH conditions in the intestinal tract and the intracellular environment to cause infection. In this study, we show that the enteric fever serovar Salmonella Typhi exhibits impaired growth at pH 4.5, in comparison to Salmonella Typhimurium. We further show that the loss of specific membrane proteins, a transcriptional regulator, and a family of stress response proteins in Salmonella Typhi are responsible for this difference. Collectively, these observations suggest that Salmonella Typhi has evolutionarily lost the ability to withstand acid stress due to differences in its interaction with the human host. This has important implications for the pathogenesis of typhoid fever.

The mycobacterial cell envelope consists of multiple covalently linked layers that must be synthesized in a coordinated manner to maintain cell wall integrity. Despite the importance of this coordination, its molecular mechanisms remain poorly understood. PgfA (polar growth factor A) interacts with trehalose monomycolate lipids (TMMs) (1) and the TMM transporter MmpL3 (1, 2). PgfA promotes TMM transport in the periplasm and functions as an upstream regulator of polar growth. How TMM transport is linked to the expansion of the entire multi-layered cell wall is unclear. Here, we provide evidence that PgfA regulates peptidoglycan metabolism. We show that PgfA localization correlates with peptidoglycan metabolism and that PgfA can function as both an activator and inhibitor of peptidoglycan metabolism. We further explore the role of TMMs in polar growth and find evidence that periplasmic TMMs are a signaling molecule that may regulate polar peptidoglycan metabolism. Finally, we find an epistatic connection between PgfA overexpression and altered TMM levels that suggests that PgfA and TMMs work in the same pathway to regulate peptidoglycan metabolism. Our data are consistent with a model in which TMM-free PgfA inhibits peptidoglycan metabolism, while TMM-bound PgfA promotes polar peptidoglycan metabolism. This work identifies PgfA as a key protein that coordinates synthesis of the peptidoglycan and mycolic acid envelope layers. ImportanceThe mycobacterial cell envelope consists of multiple covalently linked layers whose synthesis must be coordinated to maintain cell integrity. Despite decades of research on individual envelope components, the molecular mechanisms coordinating synthesis of different layers remain poorly understood. Here, we identify PgfA as a key regulatory protein that coordinates peptidoglycan and mycolate synthesis in mycobacteria. PgfA has both inhibitory and stimulatory effects on peptidoglycan metabolism, depending on the context. Our findings suggest PgfA may act as a regulator that senses mycolate precursor availability and prevents envelope imbalance when these precursors are limiting. This work provides new insight into how mycobacteria coordinate the synthesis of their complex cell envelope, with implications for better understanding mycobacterial physiology and developing antimycobacterial therapeutics.

Coxiella burnetii is the only member of the order Legionellales known to primarily infect vertebrates. The Q fever pathogen is also unusual in that it replicates within an acidified phagolysosome-like vacuole. The evolutionary origins of the virulence determinants underlying this lifestyle remain unclear. More broadly, little is known about how virulence-related traits arise in specialized intracellular lineages, where access to foreign-origin DNA may be more episodic. To address this question, we used Legionellales-wide comparative phylogenomics to reconstruct the gain and loss of traits affecting host interaction, immune evasion, intracellular survival, and metabolism. We found that many virulence-associated traits in C. burnetii predate the modern pathogen and were assembled stepwise in ancestors that likely occupied niches distinct from the acidified vacuolar niche of modern C. burnetii. The common ancestor shared with soft-tick Coxiella endosymbionts likely encoded most C. burnetii type IVB secretion system effectors, indicating that much of the host-manipulation repertoire in C. burnetii was already present before the emergence of the modern pathogen. Distinctive lipopolysaccharide features associated with immune evasion also appear to have accumulated progressively within the Coxiella lineage, including genes implicated in synthesis of virenose, a unique O-antigen sugar critical for C. burnetii virulence. Traits likely to support replication in the acidic Coxiella-containing vacuole likewise accumulated gradually, with generalized stress-tolerance functions predating acquisition of an Mrp cation/proton antiporter that may have further supported pH homeostasis. Additional changes in sugar transport and catabolism, glycolytic control, and respiratory metabolism likely enhanced metabolic flexibility and access to diverse substrates in this nutrient-rich niche. Together, these findings support a model in which vertebrate pathogenicity in C. burnetii emerged through stepwise remodeling of an ancestral host-associated lineage and provide a framework for understanding how virulence-related traits evolve in specialized intracellular pathogens. AUTHOR SUMMARYCoxiella burnetii is the bacterium that causes Q fever, a disease that can spread from animals to humans. Unlike its close relatives, C. burnetii primarily infects vertebrates and grows inside an acidic compartment within host cells. New bacterial pathogens often evolve by gaining genes from other bacteria, but how virulence evolves in lineages that grow only inside host cells, where opportunities to gain new genes may be infrequent, remains unclear. We wanted to understand how C. burnetii evolved the traits needed for its distinctive intracellular lifestyle. By comparing its genome to those of related bacteria across the order Legionellales, we found that features involved in host manipulation, immune evasion, acid tolerance, and nutrient use appeared at different times in its ancestry rather than being acquired all at once by the modern pathogen. Our findings suggest that specialized intracellular pathogens can emerge through gradual changes in ancestral host-associated lineages, including gene acquisition, gene loss, retention of older traits, and repurposing of existing functions.

Cryptosporidium is a protozoan that infects epithelial cells of the small intestine and is a cause of diarrhea and death in immunocompromised individuals and malnourished children. Immunity to this parasite is mediated by an intestinal T cell response, which is generated in gut-associated lymphoid tissues and dependent on type 1 conventional dendritic cells (cDC1s). The initial priming of T cells is accompanied by changes in integrin expression and subsequent trafficking to the site of infection. The role of specific integrins in trafficking to the ileum during cryptosporidiosis is largely unknown. The development of a transgenic Cryptosporidium strain that expresses MHCI and MHCII-restricted model antigens provides the ability to track T cell responses to this parasite. Our studies in this system revealed marked changes in the integrin profile of parasite-specific T cells as they are activated and traffic to the gut, and demonstrate that cDC1s contribute to the expression of the integrins 4, {beta}7, {beta}1, and L. Surprisingly, blockade of the canonical gut-homing integrin 4{beta}7 does not impact the ability of parasite-specific T cells to access the gut. However, blockade of integrin L decreases the parasite-specific T cell frequency at the site of infection and delays control of parasite burden. These datasets highlight an 4{beta}7-independent mechanism of T cell trafficking to the small intestine and indicate that L is an integrin required for T cell-mediated resistance to Cryptosporidium.