Xin-Ru Wang works in tick biology and intracellular bacterial pathogenesis. In this mSphere of Influence article, she reflects on how "Gut microbiota of the tick vector Ixodes scapularis modulate colonization of the Lyme disease spirochete" by Narasimhan et al. (2014) reshaped her understanding of vector competence by placing pathogen colonization within the ecological context of the tick microbiome. Her laboratory studies Rickettsia-tick cell interactions, including autophagy, apoptosis, and innate immune signaling. Here, she examines how this ecological framework extends to the intracellular level, where pathogenic Rickettsia may encounter cellular environments already shaped by resident endosymbionts.

Streptococcus pneumoniae is a gram-positive bacterium, colonizer of the human nasopharynx capable of causing severe invasive disease. Colonization of the nasopharynx is a prerequisite for the development of invasive disease and depends upon surface-bound bacterial proteins interacting with host cell receptors. Pneumococcal surface adhesin A (PsaA) is a highly conserved lipoprotein involved in the attachment to host airway cells via the host receptor Annexin A2 (ANXA2). However, the specific structural domains of PsaA responsible for ANXA2 recognition and binding remain unknown. Here, we employed a structure-based peptide approach to map the ANXA2-binding domain of PsaA. Five recombinant PsaA peptides were designed and generated based on the crystal structure of PsaA. Far-western blot analysis using ANXA2-overexpressing HEK293T/17 cell lysates revealed that the interaction with ANXA2 was specific to the C-terminal subdomain of PsaA. Cell-binding assays and flow cytometry further confirmed the interaction between the C-terminal subdomain of PsaA and cell surface-expressed ANXA2. Furthermore, polyclonal antibodies against the C-terminal subdomain significantly inhibited the binding of full-length PsaA to ANXA2-overexpressing cells, whereas antibodies against other subdomains did not. Consistently, polyclonal antibodies against the C-terminus of PsaA reduced the binding of S. pneumoniae to A549 lung epithelial cells to a greater extent than other antibodies. Together, these findings establish that the C-terminus of PsaA is crucial for ANXA2 receptor recognition. Targeting this specific subdomain may be a promising strategy for developing next-generation protein-based pneumococcal vaccines that aim at blocking bacterial adherence.IMPORTANCEStreptococcus pneumoniae is a leading cause of millions of deaths worldwide each year due to its ability to transition from an asymptomatic colonizer to an invasive pathogen. Current pneumococcal conjugate and polysaccharide vaccines protect against pneumococcal disease, but overall colonization rates have remained stable. Since pneumococcus is an opportunistic pathogen, decreasing overall colonization rates is essential for preventing progression to disease. The significance of our research lies in mapping functional epitopes within key pneumococcal adhesins that play a critical role in bacterial adherence. Defining these adhesion epitopes is essential for the rational design of next-generation protein-based vaccines capable of blocking colonization and ultimately reducing the global burden of invasive pneumococcal diseases.

Understanding whether and how microbial communities are altered by plant invasion provides important information about the impact of introduced species on natural resources, nutrient cycling, and biodiversity that influence subsequent land management and ecosystem restoration decisions. We document biotic homogenization of resident soil microbes across geographically disparate locations following a relatively recent plant invasion. We further provide evidence suggesting microbial community changes are linked to the enrichment of specific taxa from the invasive plant's rhizosphere and possible buffering of these communities against other environmental selective pressures.

Trypanosoma brucei is a model unicellular parasite for cellular and molecular genetic studies, but tools for more multiplexed experiments are limited. Tetracycline (Tet)-inducible gene regulation has been a long-standing and effective approach for overexpression, RNAi, and genome-wide screens and has been foundational for studying essential genes that are required for biological processes and identifying potential drug targets. To achieve greater flexibility in experimental design, we capitalized upon previously described dual inducer systems that combined vanillic acid (Van) or cumate (Cym) with Tet as inducers. Here, we report the development of a triple inducible system combining Cym, Van, and Tet repressors to selectively regulate the expression of three genes within a single cell line called PHITER. To demonstrate independent control, we adapted the previously characterized Van/Tet dual inducible RNAi complementation cell line, IBCompVaT, for additional Cym inducible expression of an eGFP reporter. We provide evidence that each inducer operates independently with no evidence of leaky expression. In this system, Cym induction is specific and tunable. As proof of principle, we used triple induction for POLIB RNAi, complementation with a recoded ectopic POLIB-PTP variant, and overexpression of the maxicircle helicase. We confirmed that Cym inducible PIF2 overexpression resulted in massive overreplication of maxicircles that is suppressed when POLIB is silenced, thereby demonstrating a requirement for POLIB during replication stress. The triple inducible system enables the study of complex and pleiotropic phenotypes through a more sophisticated experimental design that could not previously be achieved and can be applied for the development of multiplexed genomic screens.IMPORTANCETrypanosoma brucei is a protist parasite that causes significant health and economic burden for sub-Saharan Africa and serves as a key model organism for a group of eukaryotes called the kinetoplastids. T. brucei and related parasites contain an interlocked mtDNA network called kinetoplast DNA (kDNA), composed of maxicircles and minicircles. Although T. brucei is highly genetically tractable, limitations exist for more complex experimental designs and multiplexed forward genetic screens. Here, we describe the first triple inducible system in T. brucei or any eukaryote that combines three independent repressors (cumate [Cym], tetracycline [Tet], and vanillic acid [Van]) to selectively regulate three genes within a single cell line. We demonstrate the utility of the system using an eGFP reporter and robust RNAi complementation of kDNA polymerase POLIB. Furthermore, we investigate the role of POLIB in maxicircle replication by overexpressing the maxicircle helicase during POLIB RNAi, revealing an early requirement for POLIB during replication stress.

Utilizing treatment strategies based on collateral sensitivity (CS) represents a promising approach to suppressing antibiotic resistance. Although the mechanism of CS between numerous drugs has been researched, the mechanism of CS between tigecycline and colistin remains unclear. Therefore, the purpose of our research is to investigate the possible mechanism by which tigecycline affects colistin CS in the Enterobacter cloacae complex. Tigecycline induction significantly reduced the minimum inhibitory concentration of carbapenem-resistant Enterobacter cloacae complex (CRECC) to colistin, and sequencing revealed a single-base deletion at the RamR binding site. Complementation experiments demonstrated that deletion of the RamR binding site increased the resistance of CRECC417 to colistin and tigecycline by 2-fold and 4-fold, respectively. Transcriptomic comparison analysis of strains before and after CRECC417 induction revealed a total of 1,977 genes with significant differences in expression. Genes associated with carbohydrate, amino acid, and inorganic ion metabolism were the most highly enriched. Furthermore, the observed increase in colistin susceptibility in CRECC417R can be attributed to the inhibition of quorum sensing and biofilm formation pathways, as well as increased expression levels of genes associated with lipopolysaccharide biosynthesis and modification.IMPORTANCEDue to the overuse of antibiotics, antimicrobial resistance (AMR) has become a serious threat to global public health. Dosage regimens based on bacterial CS can reduce antibiotic use without reducing efficacy, thereby reducing antibiotic-related toxicity risks, expanding the scope of antibiotic application, and limiting the development of antibiotic resistance. In this study, we analyzed the drug resistance mutations and global transcriptional changes in CRECC after tigecycline induction through genomics and transcriptomics. Our study showed that tigecycline exposure significantly inhibited quorum sensing pathways and biofilm formation. There were significant changes in the transcriptional levels of genes related to cell membrane lipopolysaccharide synthesis and modification, but no mutations were found in genes related to colistin resistance. These findings provide valuable insights for further investigation into the CS between tigecycline and colistin.

Burkholderia pseudomallei, which causes melioidosis, poses a serious threat to human and animal health in tropical and subtropical regions worldwide. Classified as a tier 1 biothreat agent by the U.S. CDC and a category II pathogen in China, B. pseudomallei causes severe pneumonia and septicemia with case-fatality rates approaching 50%. Despite its medical and epidemiological significance, the regulatory mechanisms controlling its virulence and environmental persistence remain poorly understood. This study identifies IrlS2-IrlR2 as a previously uncharacterized two-component system (TCS) that acts as a global regulator integrating biofilm formation, stress adaptation, and virulence. Functional and transcriptomic analyses reveal that IrlS2-IrlR2 modulates secretion systems, iron homeostasis, and redox balance. These findings deepen our understanding of B. pseudomallei pathogenesis and highlight the role of TCS-mediated regulatory networks.

Mass drug administration (MDA) of azithromycin (AZ) and case finding and treatment of children with cutaneous ulcers (CUs) have been trialed as strategies to eliminate yaws. Metagenomic sequencing of CU swabs obtained from children 3 to 4 years after the initiation of a yaws elimination campaign on Lihir Island showed enrichment for Treponema pallidum subsp. pertenue (TPE), Haemophilus ducreyi (HD), Streptococcus pyogenes (SP), and several anaerobic bacteria. Whether these results were influenced by AZ pressure or are generalizable to other populations is unknown. Here, we performed quantitative PCR (qPCR) for TPE, HD, and SP DNAs and metagenomic sequencing on 260 CU specimens collected from children on New Britain Island, whose inhabitants had not received MDA of AZ. Based on qPCR results, specimens were classified as containing no pathogens, a single pathogen, or multiple pathogens. 31.9% of the specimens contained SP, 28.1% HD, and 25.4% TPE DNAs; mixed infections with two or three pathogen DNAs occurred in 16.5% of cases. No pathogen DNA was detected in 31.5% of the specimens, which were defined as idiopathic ulcers (IUs). In most categories, the same pathogen(s) used to classify the ulcers by qPCR were the most abundant taxa present in the specimens. In IU, the most abundant taxon was Arcanobacterium haemolyticum; however, its relative abundance was similar across all groups, suggesting that this organism may adapt to the CU environment rather than represent a cause of IU. These data indicate that TPE, HD, and SP are the primary causes of CU in this untreated population.IMPORTANCECutaneous ulcers (CUs) affect ~100,000 children annually in tropical regions. After mass drug administration (MDA) of azithromycin (AZ) failed to eradicate yaws, the World Health Organization proposed an integrated disease management strategy to control CU, which emphasizes identifying the causative pathogens in each population. This is critical because organisms associated with CU vary geographically, with Treponema pallidum subsp. pertenue (TPE), Haemophilus ducreyi (HD), Streptococcus pyogenes (SP), and Leishmania spp. predominating in different countries. We previously found that TPE, HD, and SP DNAs were enriched in CU specimens from children on Lihir Island in Papua New Guinea (PNG), a population heavily exposed to AZ. Here, we show that these three organisms were also the major pathogens in CU specimens from children on New Britain Island in PNG, whose population had not received MDA of AZ, suggesting that our previous findings are generalizable within PNG and not a consequence of AZ exposure.

Patients with chronic kidney disease (CKD) frequently experience constipation, often linked to gut microbiota disruptions. We hypothesized that constipation is not merely a comorbidity but a driver of adverse outcomes through a specific host-microbial metabolic axis. Employing a multi-cohort design for evidence triangulation, we first employed a large U.S. database and mortality data up to 31 December 2019 to investigate the association between constipation and overall mortality in patients with CKD using weighted Cox proportional hazards models. To explore potential microbial and metabolic mechanisms that could underlie such clinical observations, we then used genetic data from the independent FINRISK cohort to investigate causal relationships between specific gut microbiota, their metabolites, and CKD. In the observational cohort, we identified functional constipation as a potent, independent predictor of both all-cause (HR: 1.33; 95% CI: 1.11-1.58) and cardiovascular mortality. This association remained robust across sensitivity analyses and showed a distinct U-shaped dose-response relationship with stool frequency. In the causal inference phase, while the direct causal effect of constipation on CKD was not significant, MR identified a significant causal depletion of the genus Herbidospora driven by constipation. Further analyses revealed that Herbidospora exerts a protective effect against CKD. Crucially, mediation analysis demonstrated that phosphatidylcholine (14:0_18:2) [PC 14:0_18:2] metabolites mediate 12.5% of the protective pathway from Herbidospora to CKD. Constipation is a key and independent predictor of mortality in patients with CKD. We found that constipation leads to a reduction in the number of Herbidospora, thereby disrupting the protective function of the kidney. This is because PC (14:0_18:2) is lacking. This study emphasizes the importance of restoring this microbial metabolic axis as a novel therapeutic strategy for CKD.IMPORTANCEFor millions living with chronic kidney disease (CKD), a common issue such as constipation can be a hidden danger, increasing their mortality risk by over one-third. Our research uncovers why: an unhealthy gut, often indicated by constipation, lacks specific "good"" bacteria essential for producing a protective fat molecule. This natural molecule acts as a key, activating the kidney's own defense and repair systems. This discovery of a "gut-lipid-kidney" connection offers a groundbreaking new strategy: therapies aimed at restoring gut health and supplementing this key protective fat could provide a powerful new way to slow disease progression and improve survival in CKD patients.

The study of the human microbiome (mirroring broader practice across biomedical science) has historically defaulted to the use of simplified, socially constructed "boxes," such as racial and ethnic labels, that fail to accurately capture human variation and fundamentally misdirect the search for mechanisms to explain differences in health outcomes. Five years ago, I proposed a "frameshift," a fundamental conceptual shift away from relying on these categories and toward a more nuanced, careful approach to the complexity of human variation. Moving "out of the box" means tackling the difficult but essential work of analyzing microbial variation through a systems lens, connecting large-scale ecosocial drivers to individual mechanisms and outcomes. In this Full Circle review, I discuss rapid progress in the field toward this new framework and argue that by adopting transdisciplinary methods, we can generate more accurate, actionable, and equitable solutions for human health.

The increase in the infection caused by Acinetobacter baumannii is sustained by the selection of distinct epidemic clonal lineages, which are frequently resistant to a broad range of antimicrobials and possess virulence traits responsible for their persistence in the contaminated environment and spread among patients. The present study aimed to perform an integrated genomic and phenotypic analysis to assess the virulence features of ST25 isolates. A. baumannii isolates assigned to the ST25 epidemic clonal lineage shared high genomic similarity and clustered in four clades (I, II, III, and IV), with clade IV further subdivided into CIVa, CIVb, CIVc, and CIVd. Capsular locus (KL) KL14 was the predominant KL type (47%). Accessory genome analysis showed the presence of tartrate metabolism genes only in CII genomes. CIVb and CIVd ST25 A. baumannii isolates showed higher ability to infect Galleria mellonella larvae than CI, CII, CIII, and CIVc isolates. Hydrogen peroxide resistance was higher in CI, CII, CIVb, and CIVd isolates compared with CIII and CIVc isolates. In desiccation survival tests, CIII, CIVb, and CIVd isolates exhibited prolonged survival. In addition, CI, CII, CIII, CIVb, and CIVd isolates showed higher serum resistance than CIVc isolates. Also, KL14 type and lipooligosaccharide outer core locus (OCL) OCL6 type isolates were significantly more resistant to oxidative stress, to desiccation, and possessed a high ability to kill G. mellonella larvae. A positive and significant correlation was found between AdeB and AdeJ efflux pump expression and hydrogen peroxide resistance.IMPORTANCEIn this study, we characterized the genotypic and phenotypic features of A. baumannii strains assigned to the ST25 epidemic clonal lineage, which were isolated from humans, animals, and the environment. We found that ST25 A. baumannii isolates, irrespective of their antimicrobial resistance, showed peculiar virulence features among clades, isolates assigned to clade IVb and IVd showing the highest virulence and elevated resistance to serum and desiccation. Also, a positive significant correlation was found between the presence of KL14 and outer core locus 6 genotypes and resistance to oxidative stress, resistance to desiccation, and the ability to kill G. mellonella larvae. Phenotypic differences reflected clade identity rather than isolate origin, suggesting that specific virulence traits contribute to the environmental persistence and pathogenic potential of A. baumannii ST25 isolates.