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Nucleoid-associated proteins (NAPs) are essential in bacteria for maintaining nucleoid architecture and regulating the expression of target genes. Although some NAPs have been well studied in certain bacterial species, their specific functions and regulatory mechanisms remain poorly characterized in mycobacteria. In this study, we identified NapR as a novel nucleoid-associated protein in mycobacteria. We showed that NapR, which is highly conserved among mycobacteria, binds DNA and modulates DNA topology through bridging. Furthermore, we demonstrated that NapR acts as a positive transcriptional regulator of the ggr gene, which encodes geranylgeranyl reductase, thus regulating bacterial antioxidant defense. By controlling ggr expression, NapR modulates the level of intracellular ROS and influences the antioxidant capacity of mycobacteria. This study identifies NapR as a novel nucleoid-associated protein and defines a specific regulatory pathway involved in mycobacterial antioxidant defense, providing new insights into the mechanisms of the bacterial oxidative stress response.IMPORTANCEAs important global regulatory factors, nucleoid-associated proteins (NAPs) can help bacteria adapt to environmental stress, such as oxidative stress. However, the regulatory mechanism of NAPs in mycobacterial antioxidant defense is largely unclear and remains to be explored. Here, we identify NapR as a novel nucleoid-associated protein that modulates DNA topology by bridging. We revealed the regulatory effect of NapR on mycobacterial antioxidant defense. NapR positively regulates the expression of the geranylgeranyl reductase-encoding gene ggr. In addition, the ability of NapR to regulate the levels of intracellular ROS relies on ggr, ultimately leading to the antioxidant defense of Mycobacterium smegmatis. Our findings identify a new member of the NAP family and contribute to understanding the mechanisms of bacterial antioxidant defense.

The emergence of pathogens resistant to antimicrobials has become a forefront concern for clinicians and patients alike. Antimicrobial resistance (AMR) is exacerbated by the misuse and overuse of antibiotics. Pregnant women and their infants are an important area of focus, as antibiotic use during this vulnerable period of development may generate reservoirs of AMR genes, which would contribute to future risk. Identifying the extent of antibiotic use and its association with ARG composition and persistence within this window is crucial. We sought to characterize the gut resistomes of 3-month-old infants (n = 212) and pregnant women in their third trimester (n = 99) to assess ARG burden in these populations. For a subset of women and their infants (n = 33 pairs), we explored overlap of ARG. Preliminary analyses demonstrated that pregnant women and infants had markedly different resistome communities and identified other environmental and demographic characteristics to be associated with univariate differences in infant ARG composition. When controlling for the race of the mother, infant diet, and infant antibiotic exposure since birth, delivery by cesarean section was associated with increased diversity of ARG relative to the diversity of ARG in the samples from vaginally born infants. Cesarean-born infants had increased richness of aminoglycoside ARG and increased diversity of beta-lactamase and tetracycline ARG relative to vaginally born infants. Furthermore, infants consuming any formula had increased overall richness and diversity of ARG in multivariate analyses. This study provides further insight into how diet and method of delivery are associated with resistome composition within the first 3 months of infant microbiome development.IMPORTANCEPregnancy and the first 3 months of life are vulnerable periods for antibiotic exposure and subsequent development of antimicrobial resistance (AMR). AMR is an increasingly worrisome problem for global public health. The full repertoire of AMR genes present in the gut collectively is referred to as the resistome. Herein, the associations between a variety of demographic and environmental factors, including race of the pregnant women, sex of the infant, mode of delivery, amount of breast milk consumed in infant diet, and antibiotic exposure during the first 3 months of life, with resistome composition are reported. Infants consuming any formula had a greater richness and diversity of ARG overall, and cesarean-born infants had greater diversity of ARG within their resistomes. These findings give insight into the early seeding of the infant resistome, which is crucial to understanding how the resistome develops throughout life.

Mosquito rearing optimization in laboratory conditions is crucial for both vector research and control. Although the addition of nutrients is important for Aedes aegypti development from immature stages to adult mosquitoes, little is known about the nutrient composition of commercial diets used for mosquito rearing and their influence on Ae. aegypti life traits. Here, we evaluated the influence of four commercial diets commonly used to rear Ae. aegypti in the laboratory on its fitness, lifespan, and microbiota. We also compared the effect of these diets on this mosquito when combined with two different rearing waters (laboratory versus field-collected waters). Our investigations demonstrated that higher levels of protein and lipid in commercial diets promote better Ae. aegypti development, lifespan, and size in both water. Metagenomic analysis revealed specific modulations of adult microbiota composition according to both diet and rearing water. Chryseobacterium dominated the microbiota of female mosquitoes reared in laboratory water, except for yeast condition, where a more diverse microbiota was observed. When reared in larval site water, the microbiota diversity was overall higher despite diet addition, except for fish food, which promoted Sphingobacterium dominance. Given the pivotal influence of diet addition during the larval stage on Ae. aegypti microbiota and life traits, rearing conditions should be carefully chosen according to the goals of the research (i.e., vectorial capacity estimations) or vector control intervention.IMPORTANCEAedes aegypti is the main vector of arbovirus, such as dengue, yellow fever, and chikungunya viruses. Vector research and control are primarily carried out in laboratories, with larval stage rearing conducted using commercial diet. If many nutrients are essential for Ae. aegypti development, gaining insight into the influence of these diets and their nutrient levels is important to promote optimized rearing worldwide. In this study, our results indicated a significant impact of commercial diet on Ae. aegypti development, lifespan, size, and microbiota related to contrasted protein, lipid, and carbohydrate levels in these diets. This study will help people working with Ae. aegypti raise awareness in staff working with Ae. aegypti to select optimized diets for their specific purpose.

Equine strangles caused by Streptococcus equi subspecies equi (S. equi) remains a significant cause of morbidity and mortality in horses, and there is a need for improved diagnostic and vaccination strategies for addressing this pathogen. ORFeome phage display is a platform that allows for rapid screening for potential antigenic epitopes by construction of phage-displayed peptide libraries. In this study, an S. equi ORFeome library was used to screen serum from a panel of 17 horses with known exposure to S. equi to identify antigenic bacterial proteins. From this screen, three major S. equi proteins were identified: a novel proline-rich repeat domain protein, a serine peptidase, and the M-like protein SeM. These three proteins are predicted to be expressed on the surface of the bacterial cell by the presence of N- and C-terminal signals. The proline-rich repeat protein and serine peptidase were confirmed to be immunogenic by enzyme-linked immunoassay (ELISA) using the recombinant full-length proteins against sera from horses with strangles, horses infected with the related pathogen S. equi subsp. zooepidemicus, and healthy control horses. Due to the native IgG binding activity of SeM, ELISA against the full-length protein was not conducted, but the specificity of the antibody response against the recovered ORFeome clones was confirmed and an antigenic region identified. Both the proline-rich repeat protein and serine peptidase were found to be highly conserved in global S. equi genomes, indicating these proteins may be useful as vaccine candidates against S. equi or as diagnostic markers to specifically identify S. equi infections in horses.

Importance: This work utilized an ORFeome phage display platform to systematically identify antigenic epitopes produced by Streptococcus equi subspecies equi (S. equi), an important equine pathogen and the causative agent of horses strangles. Three major S. equi surface proteins were identified: a novel proline-rich repeat domain protein, a serine peptidase, and the M-like protein SeM. The proline-rich repeat protein and serine peptidase were confirmed to be immunogenic in horses with strangles, and their sequences were shown to be conserved in global S. equi genomes, in contrast to their diversity in S. equi subsp. zooepidemicus. With the well-characterized S. equi immunogenic protein SeM, this paper identified an immunogenic region outside of the reported critical IgG-binding region. This work provides novel insights to the understanding of the S. equi immunogenic proteins and provides peptide regions that could serve as vaccine candidates against S. equi or as diagnostic markers to specifically identify S. equi infections.

Candidozyma auris is a growing public health concern, capable of causing long-term contamination of healthcare settings, skin colonization, and life-threatening bloodstream infections. However, C. auris pathogenesis is not well understood, which is exacerbated by limitations and discrepancies in existing animal infection models. Further, the effects of C. auris growth phase on virulence have not been examined, despite growth phase being linked to virulence in many bacterial species. To address this question, and to develop an immunocompetent murine model of infection, we directly compared log-phase and stationary-phase C. auris systemic infection in immunocompetent C57BL/6J mice at high and low doses of infection. Systemic infection with high-dose log-phase C. auris results in rapid mortality between 2 h and 1 day post-infection, whereas stationary phase C. auris results in significantly extended survival. However, at low doses of infection, there was no difference in mortality kinetics between log-phase and stationary-phase cells. We observed that C. auris initially colonizes multiple organs but is rapidly cleared from the lungs and spleen, while kidney fungal burdens remain stable. Mice infected with high-dose log-phase C. auris had fibrin-associated blood clotting in multiple organs and decreased serum fibrinogen levels, suggesting that coagulation may drive rapid mortality. This was associated with increased β-glucan exposure and mannan abundance in log-phase C. auris. These results will inform the development of a more standardized animal model of systemic C. auris infection, which can be used to reveal key aspects of C. auris pathogenesis.IMPORTANCEDespite its growing medical importance, there is limited understanding of Candidozyma auris pathogenesis, due in part to limitations of existing laboratory models of infection. To develop a more complete understanding of factors that contribute to C. auris pathogenesis, it will be necessary to establish consistent parameters for animal models of infection. To address this need, we directly compared log and stationary growth phases on C. auris pathogenesis in immunocompetent C57BL/6J mice using a single virulent Clade I isolate. At a high dose of infection, host survival was dramatically different between log-phase or stationary-phase C. auris, suggesting that growth phase can affect C. auris pathogenesis. These differences correlated with increased exposure of pathogen-associated molecular patterns in the C. auris cell wall in log-phase cells. These results will be instrumental in the future development of standardized animal models to study C. auris pathogenesis.

Amid the antibiotic resistance crisis, biocides, including antiseptics and disinfectants, are indispensable tools to limit the spread of nosocomial infections and extensively drug-resistant bacteria. Thus, it is crucial that they remain effective. Extruding biocides from the cell using efflux systems is one mechanism by which bacteria can survive in their presence. The CepA protein from Klebsiella pneumoniae belongs to a family of heavy metal exporters but has been reported to be associated with chlorhexidine resistance, a common biocide. This study aims to elucidate the function of CepA as a biocide transporter and clarify its substrate profile in Escherichia coli. The results support the role of CepA in the transport of Fe²⁺, Mn²⁺, and possibly Cu²⁺ and Zn²⁺ to a lesser extent. The transporter also contributes to cell tolerance to iron and manganese stress. However, in contrast to previous reports, no significant impact of the expression of this protein on bacterial resistance to quaternary ammonium biocides was detected. Therefore, we propose that the main role of K. pneumoniae CepA is as a heavy metal transporter.

Importance: Understanding the structure and function of efflux pumps is crucial for addressing efflux-mediated resistance. Transporters that pump out biocides or heavy metals are not uncommon. However, to date, no transport protein capable of extruding both types of substrates has been reported. Initially, it was proposed that Klebsiella pneumoniae CepA was associated with chlorhexidine resistance and subsequently considered a biocide efflux pump. That notion also hinted at a novel group of drug efflux pumps that can extrude both biocides and heavy metals, a direct mechanism of cross-resistance between the two. The findings of this study indicate that it is more plausible that K. pneumoniae CepA is involved in metal homeostasis rather than in chlorhexidine biocide resistance in the recombinant Escherichia coli.

Edwardsiella piscicida is a Gram-negative, intracellular, enteric pathogen that primarily causes hemorrhagic septicemia in fish. It survives and replicates within host cells by delivering a subset of effector proteins via the type III secretion system (T3SS). Previous research has identified a novel T3SS effector, EseQ, in E. piscicida; however, its function remains unclear. This study reveals that EseQ binds to both α-tubulin and GEF-H1 (Rho guanine nucleotide exchange factor 1), causing microtubule destabilization and the release of activated GEF-H1. Active GEF-H1 then stimulates the conversion of GDP-RhoA (the inactive form) to GTP-RhoA (the active form), which subsequently induces stress fiber formation in a ROCK-dependent manner. Stress fibers induced by EseQ alter the architecture of zonula occludens-1-mediated intercellular junctions. This leads to increased permeability of the epithelial barrier, thereby facilitating the translocation of E. piscicida through epithelial cell layers and its invasion into zebrafish larvae. In conclusion, this study demonstrates that the Edwardsiella T3SS effector protein EseQ promotes invasion by manipulating the microtubule and actin cytoskeletons and by disrupting the epithelial barrier.IMPORTANCEEdwardsiella piscicida causes severe hemorrhagic septicemia in marine and freshwater fish worldwide, resulting in significant economic losses for the aquaculture industry (K. Y. Leung, Q. Wang, Z. Yang, and B. A. Siame, Virulence 10:555-567, 2019, https://doi.org/10.1080/21505594.2019.1621648). Our previous research identified a novel type III secretion system effector, EseQ, in E. piscicida whose function remains to be elucidated. In this work, we showed that EseQ binds to tubulin and GEF-H1 and destabilizes microtubules. GEF-H1 released from microtubules activates the RhoA-ROCK-MLCII signaling pathway, leading to stress fiber formation in epithelial cells. EseQ deforms the epithelial barrier and promotes E. piscicida's invasion in a stress fiber-dependent manner. This work contributes to the understanding of the mechanism by which E. piscicida invades host cells.