Biology subjects

Balakhmet, A.

Publications and source records attributed to Balakhmet, A..

6 recordsLinked to original sources

bioRxiv2026

An operon encoding two secreted nucleases mediates virulence in Methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen that colonizes a significant proportion of humans, contains numerous virulence factors promoting infection, and continues to threaten human lives and burden healthcare systems globally. Many MRSA virulence factors are known to be either secreted or anchored on the outer leaflet of the cell surface. Although many virulence factors have been studied intensively in MRSA, there remains a significant proportion of secreted and surface proteins that are unstudied for their potential as virulence factors. We began with identifying proteins secreted from MRSA in axenic culture using an unbiased mass-spectrometry based approach. 2 secreted proteins thus identified mapped to an operon of 6 genes, SAUSA300_1739 to SAUSA300_1744. Mutation of each of the individual genes in the operon resulted in attenuation in a mouse model of subcutaneous infection. We demonstrate that two genes in the operon, SAUSA300_1739, and SAUSA300_1740, encode nucleases with DNase activity. Genetic analysis of the SAUSA300_1739 to SAUSA300_1744 operon across several Staphylococcus aureus strains indicate that the operon is highly conserved, highlighting its importance for virulence.

microbiology↗

bioRxiv2026

Host control of Mycobacterium tuberculosis infection is not influenced by the gut microbiome

Tuberculosis (TB) is a life-threatening disease with heterogenous presentation. Approximately one-quarter of the global population is infected with Mycobacterium tuberculosis (Mtb), yet a much smaller fraction develops active TB disease. Host genetics, immune system function, and environmental factors have all been implicated in susceptibility to Mtb, yet no one factor fully explains TB heterogeneity. Strikingly, many of these same factors are linked to gut microbiome composition, which is intimately linked to systemic development of the immune system. Antibiotic treated mouse models suggest that increased gut microbiota diversity is protective against Mtb infection. In contrast, Helicobacter hepaticus colonization is correlated with exacerbated Mtb burden. However, antibiotics can have both microbial and nonmicrobial targets and studies to date have not deconvoluted these effects. Focused testing of specific microbiome members has been impossible without a gnotobiotic model for Mtb. Here, we develop the first gnotobiotic mouse model for Mtb infection and test how microbial diversity in the gut microbiome impacts host susceptibility to Mtb. Surprisingly, after intranasal challenge with Mtb, germ-free wild type mice had no difference in lung burden when compared to mice born with either a defined gut microbiome community (OMM-12) or a diverse, conventional microbiota. H. hepaticus gut colonization of OMM-12 and conventional mice also did not impact Mtb burden in the lungs in this controlled setting. H. hepaticus colonization of the gut did perturb lung immune responses associated with TB infection control. CD4+ T cells were decreased, CD8+ T cells were increased, and IL-6 production was decreased. While the gut microbiome may yet play a role in immune compromised mouse models or human disease, comparing drastically different gut microbiotas in gnotobiotically controlled C57BL/6 mice did not yield any evidence of alteration in Mtb lung burden. The hosts unique immune response to Mtb may in part make the pathogen resistant to immune disruption caused by gut microbiome changes.

microbiology↗

bioRxiv2026

Enhancement of a STING Agonist Vaccine for Tuberculosis Using Locally Supercharged MS2 Viral Capsids

Mycobacterium tuberculosis (Mtb) infection kills more people worldwide than any other pathogen. While the Bacille Calmette-Guerin (BCG) vaccine for Mtb has been widely used for over a century, it provides insufficient protection to eradicate this disease. One of our labs has recently established that a protein antigen (H1) can be combined with a STING pathway agonist to achieve strong protection against Mtb in mice, with performance that exceeds that of the BCG vaccine. However, its reliance on a synthetic cyclic dinucleotide (CDN) with relatively poor cell uptake requires higher dosing levels, thus increasing costs. To increase the efficiency of this vaccine and provide a delivery strategy that could also be used in humans, the H1 Mtb antigen and CDN adjuvant were conjugated to genome-free MS2 viral capsids that included cationic mutations to increase cell uptake. Specifically, the H1 antigen was conjugated to the external surface of MS2 using a tyrosinase-mediated oxidative coupling reaction, and the native STING agonist cGAMP was coupled to internal cysteine residues through a reductively cleavable disulfide linker. The resulting MS2-H1 and MS2-cGAMP conjugates were then co-delivered for three doses of vaccination in mice before exposure to Mtb. The MS2-based vaccine platform was observed to have comparable efficacy to the original H1/CDN formulation, but its enhanced uptake properties enabled 57-fold less CDN and 3-fold less H1 antigen. Additionally, this vaccine elicited immune responses that have been previously demonstrated to correlate with protection. The ability of the capsid shells to protect the CDN cargo during transport allowed enzymatically produced, and thus readily accessible, cGAMP to be used instead of more costly CDNs that require many synthetic steps. This, combined with the reduced overall amount of CDN and H1 that was required, could lower the production costs of future vaccines substantially. Finally, the ability of the capsid-based carriers to bypass the membrane transporters for CDNs suggests that this enhanced vaccination platform is likely to exhibit improved human efficacy in future studies.

immunology↗

bioRxiv2026

Dengue virus-specific memory B cell subsets differ as a function of infection history

The four dengue virus serotypes (DENV1-4) are a major global health threat. Infection generates protective immunity over multiple exposures with different serotypes. Virus-specific memory B cells (MBCs) can contribute to lasting protection, yet their development over multiple DENV infections remains undefined. We comprehensively evaluated frequencies of nine DENV-specific B cell subsets in 58 samples from dengue cases, comparing groups with primary (1{degrees}) versus secondary (2{degrees}) DENV infection history. Longitudinal sampling from acute infection to 18 months post-symptom onset enabled assessment of DENV-specific MBC temporal dynamics. Critically, we found that DENV-specific B cell frequency differed substantially at the level of phenotypic subsets in 1{degrees} versus 2{degrees} immunity, despite no significant difference in total frequency of DENV-specific B cells. In particular, DENV-specific IgG+, IgM+, atypical, and class-switched IgD-MBCs were durable until 18 months and accumulated with multiple exposures, representing a bona fide memory compartment against DENV. Also, naive-like IgD+/IgM+ DENV-specific B cells were found. Interestingly, the peak of certain DENV-specific MBCs occurred >3-months post-symptom onset in 2{degrees} DENV immunity, suggesting potential for long-term MBC maturation. We demonstrate that DENV-specific MBC subsets differ as a function of infection history, suggesting that 2{degrees} DENV immunity does not simply generate a quantitative boost, but a qualitative reprogramming of the memory pool. Significance StatementThe four dengue virus serotypes (DENV1-4) cause the most prevalent human mosquito-borne viral disease. Dengue is a febrile disease that often results in debilitating body pain and can rapidly progress to severe disease involving shock. People are generally protected after multiple exposures to different serotypes. Memory B cells (MBCs) can contribute to lasting protection against subsequent dengue. To understand how these rare DENV-specific MBCs develop over multiple exposures, we compared samples from cases with primary versus secondary (i.e., multiple) DENV infections. We found that instead of a higher frequency of total DENV-specific MBCs, particular subsets of DENV-specific MBCs were higher and peaked later after multiple exposures. This suggests that a qualitative shift in DENV-specific MBCs may contribute to protective immunity.

immunology↗

bioRxiv2026

A highly conserved two-gene operon is crucial for lipoarabinomannan localization, pathogenesis, and cell envelope function in Mycobacterium abscessus

Mycobacterium abscessus is an emerging threat, causing infections that are difficult to treat due to intrinsic resistance to most antibiotics. Determinants of M. abscessus physiology and pathogenesis remain poorly understood, hampering therapeutic development. Here, we show that in M. abscessus, the lprg-mfs operon is essential for virulence in macrophages and in mice. Loss of lprg-mfs in M. abscessus causes accumulation of the glycolipid lipoarabinomannan (LAM) on the cell surface and in culture supernatant suggesting that this system participates in LAM import. This contrasts with its proposed role in M. tuberculosis where lprg-mfs has been implicated in the export of various lipids. Consistent with altered lipid distribution, the lprg-mfs mutant displays severe defects in mycomembrane permeability, fluidity, and integrity, and expression of mfs alone restores only a subset of these phenotypes, revealing a surprising uncoupling of envelope fluidity and permeability. Using a suppressor screen to further investigate factors that control the distribution of lipoarabinomannan we find that a point mutation in the unannotated gene MAB_0995 can fully or partially complement all deletion mutant phenotypes. Our data also show that lipoarabinomannan in the mycomembrane is dynamically regulated in response to environmental conditions, including hypoxia and macrophage infection. Together, these findings redefine the role of LprG/Mfs in mycobacterial cell envelope homeostasis and reveal unexpected plasticity in mycomembrane lipid regulation in M. abscessus. ImportanceThe emerging pathogen Mycobacterium abscessus causes life-threatening lung infections in certain patients that are extremely difficult to treat due to its intrinsic resistance to most antibiotics. However, the process by which this organism establishes infection is poorly understood, as are the specific determinants of antibiotic tolerance. Better knowledge of the genes required for virulence and impermeability to antibiotics in M. abscessus could enable to development of more effective treatments. The significance of this study is the demonstration that the lprg-mfs operon is required both for pathogenesis and for impermeability in M. abscessus. Further, our study shows a correlation between cell envelope characteristics and the distribution of the molecule lipoarabinomannan, suggesting a specific mechanism by which these crucial characteristics are mediated.

microbiology↗

bioRxiv2025

Mycobacterium tuberculosis suppresses protective Th17 responses during infection through multiple mechanisms

Mycobacterium tuberculosis (Mtb) causes more deaths annually than any other pathogen, yet an effective vaccine remains elusive. IFN-{gamma}-producing Th1 CD4+ T cells are necessary but insufficient for protection against infection. In humans, the development of IL-17A producing Th17 T cells correlates with protection, however not all individuals develop a Th17 response. In mice, experimental vaccines can elicit protective Th17 cells, yet Th17s are rare in primary infection. Why Mtb fails to consistently elicit Th17s is unknown. Here, we identify factors suppressing Th17 responses during primary infection. We demonstrate that the lack of Th17 induction is independent of route and duration. Next, using Tbet deficient mice, we show that Mtb drives a Th1 response that is only partially protective and limits Th17 cell production in an IFN-{gamma} independent manner. We further reveal that the ESX-1 type VII secretion system and lipid PDIM suppresses Th17 responses. Infection with ESX-1 or PDIM mutants results in significantly increased Th17 T cells and IL-17A cytokine in the lungs, and infection of IL-17A deficient animals partially restores virulence of ESX-1 and PDIM mutants. Although the ESX-1 secretion system and lipid PDIM elicits type I IFN, which can suppress Th17 differentiation, we find that suppression of Th17 is independent of type I IFN. Instead, ESX-1 and PDIM suppresses production of IL-23, a cytokine that promotes Th17 differentiation, in dendritic cells found in mediastinal lymph nodes during Mtb infection. These findings define a new function of the ESX-1 secretion system and PDIM in Mtb virulence, a long-standing question in tuberculosis research.

immunology↗