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Carcinoembryonic antigen (CEA) is a critical colorectal cancer (CRC) biomarker, but its mechanistic link to gut microbiota remains unclear. This study characterized gut microbiota differences between high-CEA (H-CEA) and low-CEA (L-CEA) CRC patients and explored their associations with host immunity and tumor progression mechanisms. Stool samples from 187 CRC patients were subjected to 16S rRNA sequencing, identifying 30 differentially abundant bacteria using LEfSe analysis. Ruminococcus callidus was significantly enriched in H-CEA patients. Transcriptome sequencing of tumor tissues from 25 patients revealed distinct immune micro-environments: H-CEA patients showed elevated resting memory CD4⁺ T cells, while L-CEA patients showed increased T follicular helper cells. Functional enrichment analysis identified differential GO terms (26 in L-CEA; 31 in H-CEA) and KEGG pathways (three in H-CEA). R. callidus correlated positively with mast cell infiltration, CXCL1 chemokine, and long-chain fatty acid upregulation. The area under the curve (AUC) values of the subjects in the training set for the RF and XGBoost models constructed based on differential gut microbiota for predicting high and low CEA levels were 0.969 and 0.815, respectively, and the AUC for the test set were 0.715 and 0.639. These findings demonstrate that CEA-level-specific gut microbiota dysbiosis modulates CRC progression through immune micro-environment alterations and related biological pathway regulation. Gut microbiota, as a noninvasive biomarker, can be used to construct an effective machine learning (ML) model for predicting blood CEA levels.

Importance: This study reveals R. callidus as a key gut microbiota species enriched in CRC patients with high CEA levels, demonstrating its novel pro-tumor associations through positive correlations with mast cell infiltration and CXCL1 chemokine and upregulation of long-chain fatty acid metabolism. Concurrently, we identify distinct immune micro-environments: elevated resting memory CD4+ T cells in high-CEA patients versus increased T follicular helper cells in low-CEA cohorts. Critically, by leveraging 30 differential microbial features, we develop ML models for noninvasive prediction of CEA levels. These findings establish gut microbiota as both a mechanistic mediator of CEA-driven CRC progression and a foundation for microbiome-based diagnostic tools.

Hsp90 is considered to be the master regulator of chaperone activity within the cellular context. In addition to aiding client maturation and maintaining protein homeostasis, Hsp90 serves various non-canonical functions in model eukaryotes: ranging from protein-trafficking into the nucleus to transcriptional regulation, from chromatin remodeling to assembly and disassembly of protein complexes during DNA repair and telomere maintenance. In performing all these trades, Hsp90 collaborates with its co-chaperones in a client-specific or function-specific manner. Hsp90 undergoes various conformational changes during its chaperone cycle, which is regulated via several post-translational modifications (PTM). Different combinations of such PTMs, known as the chaperone code, also play key regulatory roles for Hsp90 functions. Here, we examine various cellular functions of Hsp90 in protozoan parasites, particularly those that shuttle between insect host and human host, adapting to a temperature difference of at least 10°C. Our analyses reveal that most of the prominent co-chaperones are present in all these parasites, except for one that is essential in model eukaryotes. We reviewed the biochemical correlates of Hsp90 and its co-chaperone interactions and realized that the physiological significance of such interplay is largely unknown in the protozoan parasites. One striking observation is the lack of sequence conservation of the parasitic co-chaperones with their human counterparts, making them attractive drug targets. Our analyses revealed that in spite of the identification of few PTMs of parasitic Hsp90 proteins, the chaperone codes remain largely elusive.IMPORTANCEHsp90 is a pivotal molecular chaperone involved in maintaining proteostasis and facilitating the maturation of diverse client proteins. Beyond its canonical folding functions, Hsp90 plays non-canonical roles in nuclear trafficking, transcriptional regulation, chromatin remodeling, and DNA repair. These activities are tightly regulated through interactions with specific co-chaperones and through post-translational modifications, collectively forming the "chaperone code." This study examines Hsp90's role in thermal adaptation of protozoan parasites when shuttling between the insect and human hosts. Here, we summarize the canonical and diverse non-canonical functions of Hsp90 in three protozoan parasites: Plasmodium, Leishmania, and Trypanosoma. We highlight all the Hsp90 isoforms found in these three parasites and also illustrate all the co-chaperones and post-translational modifications of Hsp90 found to be present in these protozoan parasites. Importantly, the divergence in co-chaperone sequences from human homologs in these parasites presents a promising avenue for targeted antiparasitic drug discovery and development.

George Luedemann is known throughout the antimicrobial community as one of the discoverers of the natural product antibiotic gentamicin. He subsequently hypothesized that slow-growing organisms inhabiting inhospitable, nutrient-limited environments may represent an enriched source of previously undescribed microbes that produce novel antimicrobials to create a competitive advantage over faster-growing rival organisms. Accordingly, 750 slow-growing microorganisms were isolated from desert rock surfaces and archived prior to Dr. Luedemann's passing in 2000. Here, we describe the characterization and antimicrobial screening of the first 147 members of the Luedemann collection. 16S rRNA and whole-genome sequencing revealed that the pilot isolate set is highly diverse and includes novel microbial species belonging to genera commonly associated with soil samples, including Geodermatophilus, Streptomyces, and Micromonospora. Antimicrobial screening and comparative genomics indicate that at least six members are likely to produce novel antimicrobials with activity toward the ESKAPE pathogens, Vibrio cholerae and/or Mycobacterium smegmatis. Indeed, we show that the library member "9005BA" produces a newly identified phenazine, pyocyanin A, which displays potent (0.625 µg/mL), selective bactericidal activity toward Acinetobacter baumannii and efficacy in animals. Genetic and biochemical assays revealed that the antimicrobial activity of pyocyanin A is likely to be mediated by oxidative stress and can be overcome by altering bacterial respiration and/or efflux. Taken together, the data suggest that slow-growing organisms inhabiting nutrient-limited environments represent a previously overlooked rich source of microbial and antimicrobial agent diversity.IMPORTANCEThe discovery and study of novel bacterial species offer an opportunity to identify new microbial biological processes, molecular mechanisms, and secondary metabolites, such as new antibiotics. Our work indicates that slow-growing organisms inhabiting nutrient-limited environments may represent an enriched source of novel microbial species. Furthermore, we find that a subset of these organisms is likely to produce corresponding novel antimicrobials, presumably as a means to outcompete faster-growing rival organisms. Indeed, we show that a putative new Streptomyces species is capable of producing a previously undescribed antimicrobial, pyocyanin A, with potent, selective antibacterial toward Acinetobacter baumannii, a prominent cause of antibiotic-resistant infections.

Clostridioides difficile is an opportunistic gastrointestinal pathogen capable of asymptomatic colonization and causes diseases ranging from diarrhea to pseudomembranous colitis. Accurate diagnosis of C. difficile infection (CDI) is challenging and critical for treatment and control. We hypothesized that gut microbiome profiles could help distinguish C. difficile colonized patients with diarrhea from those with true CDI. We analyzed 172 stool samples from individuals who tested glutamate dehydrogenase positive for C. difficile. Participants were categorized by toxin status (i.e., toxin positive or negative) and then further classified into three toxin groups based on the production of toxin, and if not produced, whether the C. difficile strain carried toxin-encoding genes. We examined associations between patient characteristics, prior antibiotics exposure, microbiome community structure and function, and toxin categories. Thirty-five percent of toxin-negative participants received antibiotics despite not meeting the criteria for true CDI. Enterococcus species were abundant in all groups. The relative abundance of E. faecalis was higher among individuals with prior antibiotics exposure. Alpha and beta diversity did not differ by toxin group. After controlling for prior antibiotics exposure and previous CDI episode, the abundance of Akkermansia muciniphila, Flavonifractor plautii, and Bifidobacterium adolescentis distinguished individuals with toxin-positive C. difficile. C. difficile abundance did not differentiate participants with true CDI from those who were colonized. We identified associations between the gut microbiome and C. difficile toxin gene presence and toxin production. These results highlight the potential for microbiome-informed diagnostics to improve CDI accuracy and guide treatment decisions.IMPORTANCEClostridioides difficile colonizes humans and causes diarrhea in community and hospital settings. C. difficile infection (CDI) is a toxin-mediated disease, and its diagnosis is challenging. The goal of this study was to determine whether differences in the gut microbiome could help distinguish between colonized individuals and those with CDI. We examined stool samples and data from 172 individuals categorized into three groups based on the detection of toxin and, if not detected, whether toxin-encoding genes were present in the C. difficile strain. We identified bacteria, such as Enterococcus faecalis, that were more abundant in people who had used antibiotics. While the diversity of the gut microbiome did not differ by toxin group, specific gut bacteria, antibiotic resistance genes, and metabolic pathways were associated with toxin group. Our findings suggest that considering the full gut microbiome and factors like past antibiotic use could help improve the diagnosis and treatment of CDI.

Bile salts play a critical role in modulating the host gut. They possess antimicrobial properties wherein they disrupt the bacterial membrane and produce reactive oxygen species (ROS), causing DNA damage. Pathogens like Salmonella regulate their metabolic activity to counteract the effects of bile. This study investigates the role of YqhD, an aldehyde reductase, in Salmonella's bile salt susceptibility. Our findings reveal increased survival of the yqhD mutant in the in-vitro studies in LB media with bile, liver cell line HepG2 and C57BL/6 mice on treatment with 8% sodium cholate in the cecum. Bile salts, physiologically produced for the digestion of fat, enhanced the organ burden of the yqhD mutant in C57BL/6 mice on replacing the chow diet with a high-fat diet (HFD). The yqhD mutation, on bile salt exposure, also leads to increased ROS levels and modulation of antioxidant genes in the bacteria. The addition of the antioxidant glutathione during bile stress enhances the survival of STM WT and makes it similar to the survival of STM ΔyqhD. Similarly, in the gp91^-/-phox mice, the organ burden and pathology of the liver and spleen were increased on STM WT infection, while it remained similar for the yqhD mutant on exposure to HFD compared to that of chow-fed mice. Furthermore, the yqhD mutant exhibited increased AcrAB efflux pump activity, regulated by RamA/R regulon.

Importance: Foodborne pathogen Salmonella can tolerate high concentrations of bile and even survive the harsh environment of the gall bladder. This study is significant as it explores the role of a novel antioxidant gene yqhD in bile salt susceptibility of Salmonella Typhimurium and Typhi. It highlights how the presence of gene yqhD, though advantageous in macrophages, reduces the Salmonella survival on bile salt exposure in vitro and in liver cell line HepG2. Deletion of yqhD increased the survival on bile stress exposure, which was attributed to its ability to induce the AcrAB efflux pump of Salmonella. A deeper understanding of how Salmonella modulates gene expression in response to bile stress could provide valuable insights into addressing the chronic carriage of Salmonella.

Clostridioides difficile spores are essential for initiation, recurrence, and transmission of C. difficile infections (CDI). These outermost layers of the spore, the exosporium and spore coat, are responsible for initial interactions with the host and spore resistance properties, respectively. Several spore coat/exosporium extraction methods have been utilized to study the spore surface, with differing procedures making comparison across studies difficult. Here, we tested how commonly used exosporium and spore coat extraction methods, termed EBB, USD, and Laemmli, remove the spore coat and exosporium layers of C. difficile spores. We assessed the impact of these extraction methods on the spore through transmission electron microscopy, phase contrast microscopy, western blotting, and lysozyme-triggered cortex degradation. Transmission electron microscopy shows that treatment with EBB and USD completely removes the spore coat and exosporium layer while leaving decoated spores intact. Western blots revealed differences in the ability to extract spore surface protein markers (CdeC, CdeM, CotA). In addition, lysozyme was able to degrade the cortex in decoated spores regardless of the treatment employed. Western blot analysis of lysozyme-treated decoated spores reveals that EBB and USD treatment allow for the detection and release of the spore core germination protease, GPR. Our results provide a comparison of commonly used extraction methods in C. difficile spore biology, standardizing their impact on spore coat and exosporium extraction for use in future studies.

Importance: The outermost layers of Clostridioides difficile spores, the exosporium and spore coat, are essential for the spores' resistance properties and initial interactions with the host. However, there is variability in extraction protocols, making it difficult to compare across studies. This work evaluates the commonly used extraction methods EBB, USD, and Laemmli at removing the exosporium and spore coat and provides a foundation for improved reproducibility. Here, we identified the effectiveness of these different extraction methods, allowing us to better understand these techniques to accurately analyze the spore surface in C. difficile spore research.

As one of the deadliest infectious diseases in the world, tuberculosis is responsible for millions of new cases and deaths reported annually. The rise of drug-resistant tuberculosis, particularly resistance to first-line treatments like rifampicin, presents a critical challenge for global health, which complicates the treatment strategies and calls for effective diagnostic and predictive tools. In this study, we apply an ensemble-based molecular dynamics computer simulation method, TIES_PM, to estimate the binding affinity through free energy calculations and predict rifampicin resistance in RNA polymerase. By analyzing 61 mutations, including those in the rifampicin resistance-determining region, TIES_PM produces reliable results in good agreement with clinical reference and identifies abnormal data points indicating alternative mechanisms of resistance. In the future, TIES_PM is capable of identifying and selecting leads with a lower risk of resistance evolution and, for smaller proteins, it may systematically predict antibiotic resistance by analyzing all possible codon permutations. Moreover, its flexibility allows for extending predictions to other first-line drugs and drug-resistant diseases. TIES_PM provides a rapid, accurate, low-cost, and scalable supplement to current diagnostic pipelines, particularly for drug resistance screening in both research and clinical domains.IMPORTANCEAntimicrobial resistance (AMR), a global threat, challenges early diagnosis and treatment of tuberculosis (TB). This study employs TIES_PM, a free-energy calculation method, to efficiently predict AMR by quantifying how mutations in bacterial RNA polymerase (RNAP) affect rifampicin (RIF) binding. On simulating 61 clinically observed mutations, the results align with WHO classifications and reveal ambiguous cases, suggesting alternative resistance mechanisms. Each mutation requires ~5 h, offering rapid, cost-effective predictions. An ensemble approach ensures statistical robustness. TIES_PM can be extended to smaller proteins for systematic codon permutation analysis, enabling comprehensive antibiotic resistance prediction, or adapted to identify low-resistance-risk drug leads. It also applies to other TB drugs and resistant pathogens, supporting personalized therapy and global AMR surveillance. This work provides novel tools to refine resistance mutation databases and phenotypic classification standards, enhancing early diagnosis while advancing translational research and infectious disease control.

The early infant gut microbiota is generally dominated by bifidobacteria, but there is substantial variation at the (sub)species level. Patterns of postnatal Bifidobacterium subspecies colonization in low- or middle-income countries have not been widely studied. We used (sub)species-specific qPCR to quantify B. infantis (n = 1132), B. longum (n = 364), and B. breve (n = 399) in stool samples from infants (0-6 months of age) in urban Dhaka, Bangladesh. B. infantis absolute abundance started low at birth but increased in the first two months, whereas B. longum and B. breve abundances remained comparatively low. B. infantis emerged earlier in infants delivered by C-section, but by ~2 months of age, infants delivered by C-section or vaginally had similar B. infantis absolute abundances. Infant antibiotic exposure (ever vs. never), human milk feeding patterns (exclusive, predominant, and partial), and detection of maternal stool B. infantis were not associated with infant B. infantis. In settings where B. infantis is widespread, its patterns of postnatal colonization can be used to inform the design of targeted microbiota-modifying interventions in infancy.IMPORTANCEBifidobacteria are considered to be an important member of the early infant gut microbiota, but several factors may influence the timing of their emergence and overall abundance. Moreover, bifidobacteria abundance varies considerably between different species and subspecies, underscoring the importance of techniques that enable sub-speciation. B. longum subspecies infantis (B. infantis) is thought to have several health-promoting properties, and despite growing interest in the use of B. infantis to promote health (e.g., probiotics), relatively few studies have explored its natural patterns of colonization, particularly in low- and middle-income countries. By applying (sub)species-specific qPCR, we precisely tracked the timing of emergence, longitudinal abundance patterns, and ecological dynamics of B. infantis, B. longum, and B. breve in the postnatal period, which provided new insights to inform the design of targeted microbiota-modifying interventions in early infancy.