Biology subjects

Zafar, M. A.

Publications and source records attributed to Zafar, M. A..

9 recordsLinked to original sources

bioRxiv2026

Influenza A Virus Coinfection Alters Streptococcus pneumoniae Gene Expression during Upper Respiratory Tract Colonization

Streptococcus pneumoniae (Spn) asymptomatically colonizes the upper respiratory tract (URT), a niche from which it can transmit to another host or cause invasive disease in the same host. The in vivo transcriptional adaptations that Spn undergoes during nasopharyngeal colonization, particularly during influenza A virus (IAV) coinfection, are poorly understood. Here, we leveraged an established infant mouse model of colonization, shedding, and transmission to perform genome-wide transcriptomic profiling of Spn during mono- and during IAV coinfection. Compared with broth-grown controls, pneumococci isolated from the URT exhibited distinct transcriptional programs, with over 200 genes differentially expressed across time points. Genes involved in carbohydrate uptake and metabolism, glycan degradation, amino sugar and nucleotide sugar metabolism, and amino acid biosynthesis were consistently enriched during colonization, highlighting metabolic adaptation to the nasopharyngeal niche. In contrast, IAV coinfection induced a markedly distinct transcriptional signature, including upregulation of branched-chain amino acid biosynthesis, bacteriocin production, and phosphate acquisition systems. Notably, the pilus islet-1 locus was upregulated during Spn-IAV coinfection. Functional studies demonstrated that while the pilus was dispensable for colonization under mono- and coinfection conditions, it promoted high-shedding events and enhanced inflammatory responses during IAV coinfection. However, reduced inflammation and reduced high shedding events from pups inoculated with a pilus-deficient mutant did not alter transmission frequency in the infant mouse model. Collectively, our findings define the in vivo transcriptional landscape of Spn during URT colonization and reveal distinct bacterial adaptations during viral coinfection, providing insight into mechanisms that influence pneumococcal persistence, inflammation, and transmission.

microbiology↗

bioRxiv2026

DksA-Dependent Stringent Stress Response Drives Virulence and Gastrointestinal Persistence of Klebsiella pneumoniae

Successful gastrointestinal colonization (GI) by bacterial pathogens requires adaptation to nutrient competition and host-derived stresses in the gut, with adaptation via the bacterial stringent stress response playing a critical role. Epidemiological data suggest that the GI tract serves as a reservoir from where K. pneumoniae can spread and cause invasive disease or transmit to another host. DksA is a conserved stringent response transcriptional regulator that was identified in an in vivo transposon mutagenesis screen as an important K. pneumoniae gut determinant. However, its role in K. pneumoniae pathogenesis and gut colonization remains uncharacterized. Here, we demonstrate that DksA is required for survival against membrane-targeting antibiotics, consistent with a role in cell envelope stress tolerance. In addition, DksA positively regulates capsule biosynthesis gene expression and hypermucoviscosity and is essential for robust biofilm formation. Using a murine model, we show that DksA functions as a determinant of GI colonization independently of the resident gut microbiota. Furthermore, we demonstrate that DksA is important for environmental survival and transmission by regulating RpoS, thereby providing a mechanistic link between the stringent stress response, environmental survival, and subsequent transmission. Together, these findings establish DksA as a central integrator of the stringent response, coordinating membrane stress resistance, virulence traits, and gastrointestinal colonization in K. pneumoniae. ImportanceK. pneumoniae, a pathobiont, is responsible for multidrug-resistant infections and poses a major threat in hospital settings as well as community-acquired invasive infections. The bacterium tightly coordinates its virulence-associated traits to adapt to diverse environmental conditions and survive; however, the regulatory mechanisms remain poorly understood. In this study, we demonstrated that the conserved stringent response regulator DksA contributes to bacterial membrane stability, thereby affecting antibiotic resistance, inherent virulence, and persistence traits of K. pneumoniae. Additionally, DksA was identified as required for gut colonization, environmental survival through dysregulation of RpoS, and transmission to a naive host. These results enhance our overall understanding of the K. pneumoniae stringent response and will provide new avenues for controlling K. pneumoniae infections.

microbiology↗

bioRxiv2025

Glutamine-Dependent Biosynthetic Pathways Fuel Autoreactive T and B Cells in Foxp3 Deficiency-mediated Disease

Foxp3 deficiency causes a profound loss of immune tolerance, unleashing autoreactive T and B cells, lymphoproliferation, cytokine-driven inflammation, and autoantibody production. This autoimmune pathology is fueled by increased glutamine usage, but it remains unresolved whether glutamine is necessary to produce energy, or for biosynthetic pathways leading to inosine and asparagine production. Here, we demonstrate that glutamine utilization supports Foxp3-deficiency mediated disease independently of pathogenic Foxp3-deficient Treg cell energetic reprogramming. Mechanistically, glutamine biosynthetic pathways sustain conventional T cell activation and proinflammatory cytokine production preventing inosine accumulation and signaling, thus implicating adenosine pathway modulation in autoreactive T cell dysregulation. Conversely, autoreactive B cell activation and autoantibody production depend on glutamine-dependent asparagine synthesis, which we reveal as a targetable vulnerability for autoantibody formation. These findings highlight glutamine-driven biosynthetic processes as critical drivers of autoimmunity and reveal distinct metabolic vulnerabilities in autoreactive T and B cells that can be targeted for therapeutic intervention.

immunology↗

bioRxiv2025

Divergent roles for complement components C3 and C4 in controlling Klebsiella pneumoniae gut colonization and systemic dissemination

Klebsiella pneumoniae is an escalating public health threat driven by the emergence of antibiotic-resistant and hyper-encapsulated strains that spread systemically from the gut. The immune defenses preventing gut colonization and dissemination remain poorly defined. Herein, we uncover distinct and context-dependent roles for complement proteins C3 and C4 in host defense following K. pneumoniae infection. Following gut colonization, C3 and C4 levels rise significantly. In addition to inducing alternative pathway-mediated C3b deposition on K. pneumoniae grown under gut-relevant conditions, C3 is critical for recruiting myeloid cells to the gut, promoting local opsonophagocytosis, and preventing lethal systemic spread. Depletion of systemic C3 reveals mucosal-derived C3 controls K. pneumoniae GI colonization, whereas systemic C3 is essential for limiting fatal dissemination. In contrast, C4 is dispensable for controlling GI colonization, dissemination, and myeloid recruitment under conditions of natural acquisition. However, C4 becomes critical for controlling GI burden and systemic disease following antibiotic-induced dysbiosis and supercolonization with antibiotic-resistant K. pneumoniae. Notably, mice deficient in CD21/35--a receptor for cleaved C3 and C4 fragments important for B cell activation and antigen retention--exhibit a defect similar to C4-/- mice, with significantly increased GI burden under antibiotic-induced supercolonization, suggesting distinct complement-dependent pathways are involved in mucosal protection. Collectively, these findings reveal a dual-layered immune strategy: C3-driven opsonophagocytosis is critical for controlling colonization and dissemination under baseline conditions, while C4 and CD21/35 become indispensable following antibiotic-induced supercolonization. This work advances our understanding of complement-dependent mucosal immune protection and identifies potential targets for preventing gut-to-bloodstream transition of this pathogen.

immunology↗

bioRxiv2022

The two-component system YesMN promotes pneumococcal host-to-host transmission, and regulates genes involved in zinc homeostasis

The ability to sense and respond rapidly to the dynamic environment of the upper respiratory tract (URT) makes Streptococcus pneumoniae (Spn) a highly successful human pathogen. Two-component systems (TCS) of Spn sense and respond to multiple signals it encounters allowing Spn to adapt and thrive in various host sites. Spn TCS have been implicated in their ability to promote pneumococcal colonization of the URT and virulence. As the disease state can be a dead-end for a pathogen, we considered whether TCS would contribute to pneumococcal transmission. Herein, we determined the role of YesMN, an understudied TCS of Spn, and observe that YesMN contributes towards pneumococcal shedding and transmission but is not essential for colonization. The YesMN regulon includes genes involved in zinc homeostasis and glycan metabolism, which are upregulated during reduced zinc availability in a YesMN dependent fashion. Thus, we identify the YesMN regulon and the molecular signals it senses that lead to the activation of genes involved in zinc homeostasis and glycan metabolism. Furthermore, in contract to Spn mono-infection, we demonstrate that YesMN is critical for high pneumococcal density in the URT during influenza A (IAV) coinfection. We attribute reduced colonization of the yesMN mutant due to increased association with and clearance by the mucus covering the URT epithelial surface. Thus, our results highlight the dynamic interactions that occur between Spn and IAV in the URT, and the role that TCS play in modulation of these interactions.

microbiology↗

bioRxiv2022

Klebsiella pneumoniae L-Fucose metabolism promotes gastrointestinal colonization and modulates its virulence determinants

Colonization of the gastrointestinal (GI) tract by Klebsiella pneumoniae (K. pneumoniae) is generally considered asymptomatic. However, gut colonization allows K. pneumoniae to either translocate to sterile site within the same host or transmit through the fecal-oral route to another host. K. pneumoniae gut colonization is poorly understood, but knowledge of this first step toward infection and spread is critical for combatting its disease manifestations. K. pneumoniae must overcome colonization resistance (CR) provided by the host microbiota to establish itself within the gut. One such mechanism of CR is through nutrient competition. Pathogens that metabolizes a broad range of substrates have the ability to bypass nutrient competition and overcome CR. Herein, we demonstrate that in response to mucin derived fucose, the conserved fucose metabolism operon (fuc) of K. pneumoniae is upregulated in the murine gut and subsequently show that fucose metabolism promotes robust gut colonization. Growth studies using cecal filtrate as a proxy for the gut lumen illustrates the growth advantage that the fuc operon provides K. pneumoniae. We further show that fucose metabolism allows K. pneumoniae to be competitive with a commensal E. coli isolate (Nissle). However, Nissle is eventually able to out-compete K. pneumoniae, suggesting that it can be utilized to enhance CR. Lastly, we observed that fucose metabolism positively modulates hypermucoviscosity, auto-aggregation, and biofilm formation, but not capsule biogenesis. Together, these insights enhance our understanding of the role of alternative carbon sources on K. pneumoniae gut colonization and the complex relationship between metabolism and virulence in this species. ImportanceKlebsiella pneumoniae (K. pneumoniae) is a leading cause of hospital-acquired infection. Treatment of infection by K. pneumoniae isolates is becoming difficult as this pathogen becomes increasingly antibiotic resistant. While there has been extensive investigation into the disease states associated with K. pneumoniae, its colonization of the gastro-intestinal (GI) tract is poorly understood. Epidemiological data suggest that in many cases the strain that colonizes the GI tract causes disease manifestations in the same host. Herein, we used our newly developed murine model of K. pneumoniae gut colonization, where colonization is achieved without disrupting the resident gut microbiota. We demonstrate that K. pneumoniae uses fucose as an alternative carbon source present in the gut lumen to overcome the intense nutritional competition. We further illustrate that K. pneumoniae, through fucose metabolism, is initially competitive with the probiotic E coli isolate Nissle 1917 (EcN). Lastly, we show that fucose metabolism modulates several virulence determinants of K. pneumoniae. Thus, our results provide new insight into the role fucose metabolism plays in gut colonization and virulence of K. pneumoniae, and furthermore identify EcN as having the ability to out-compete K. pneumoniae and be used as a probiotic.

microbiology↗

bioRxiv2021

MgrB dependent colistin resistance in Klebsiella pneumoniae is associated with an increase in host-to-host transmission.

Due to its high transmissibility, Klebsiella pneumoniae (Kpn) is one of the leading causes of nosocomial infections. Here, we studied the biological cost of colistin resistance, an antibiotic of last resort, of this opportunistic pathogen using a murine model of gut colonization and transmission. Colistin resistance in Kpn is commonly the result of inactivation of the small regulatory protein MgrB. Without a functional MgrB, the two-component system PhoPQ is constitutively active, leading to increased lipid A modifications and subsequent colistin resistance. Using an engineered MgrB mutant, we observed that MgrB-dependent colistin resistance is not associated with a fitness defect during in vitro growth conditions. However, colistin-resistant Kpn colonizes the murine gut poorly, which may be due to the decreased production of capsular polysaccharide by the mutant. The colistin-resistant mutant of Kpn had increased survival outside the host when compared to the parental colistin-sensitive strain. We attribute this enhanced survivability to dysregulation of the PhoPQ two-component system and accumulation of the master stress regulator RpoS. The enhanced survival of the colistin resistant strain may be a key factor in the observed rapid host-to-host transmission in our model. Together, our data demonstrate that colistin-resistant Kpn experiences a biological cost in gastrointestinal colonization. However, this cost is mitigated by enhanced survival outside the host, increasing the risk of transmission. Additionally, it underscores the importance of considering the entire life cycle of a pathogen to truly determine the biological cost associated with antibiotic resistance. ImportanceThe biological cost associated with colistin resistance in Klebsiella pneumoniae (Kpn) was examined using a murine model of Kpn gut colonization and fecal-oral transmission. A common mutation resulting in colistin resistance in Kpn is a loss-of-function mutation of the small regulatory protein MgrB that regulates the two-component system PhoPQ. Even though colistin resistance in Kpn comes with a fitness defect in gut colonization, it increases bacterial survival outside the host enabling it to more effectively transmit to a new host. The enhanced survival is dependent upon the accumulation of RpoS and dysregulation of the PhoPQ. Hence, our study expands our understanding of the underlying molecular mechanism contributing to the transmission of colistin-resistant Kpn.

microbiology↗

bioRxiv2020

An Animal Model to Study Klebsiella pneumoniae Gastro-Intestinal Colonization and Host-to-Host Transmission

An important yet poorly understood facet in the life cycle of a successful pathogen is the host-to-host transmission. Hospital-acquired infections (HAI) resulting from the transmission of drug-resistant pathogens affect hundreds of millions of patients worldwide. Klebsiella pneumoniae (Kpn), a gram-negative bacterium, is notorious for causing HAI, with many of these infections difficult to treat as Kpn has become multi-drug resistant. Epidemiological studies suggest that Kpn host-to-host transmission requires close contact and generally occurs through the fecal-oral route. Herein, we describe a murine model that can be utilized to study mucosal (oropharynx and gastrointestinal [GI]) colonization, shedding within feces, and transmission of Kpn through the fecal-oral route. Using an oral route of inoculation, and fecal shedding as a marker for GI colonization, we show that Kpn can asymptomatically colonize the GI tract of immunocompetent mice, and modifies the host GI microbiota. Colonization density within the GI tract and levels of shedding in the feces differed among the clinical isolates tested. A hypervirulent Kpn isolate was able to translocate from the GI tract and cause hepatic infection that mimicked the route of human infection. Expression of the capsule was required for colonization and, in turn, robust shedding. Furthermore, Kpn carrier mice were able to transmit to uninfected cohabitating mice. Lastly, treatment with antibiotics led to changes in the host microbiota and development of a transient super-shedder phenotype, which enhanced transmission efficiency. Thus, this model can be used to determine the contribution of host and bacterial factors towards Kpn dissemination.

microbiology↗

bioRxiv2018

An infant mouse model of influenza virus transmission demonstrates the role of virus-specific shedding, humoral immunity, and sialidase expression by colonizing Streptococcus pneumoniae.

The pandemic potential of influenza A viruses (IAV) depends on the infectivity of the host, transmissibility of the virus, and susceptibility of the recipient. While virus traits supporting IAV transmission have been studied in detail using ferret and guinea pig models, there is limited understanding of host traits determining transmissibility and susceptibility because current animal models of transmission are not sufficiently tractable. Although mice remain the primary model to study IAV immunity and pathogenesis, the efficiency of IAV transmission in adult mice has been inconsistent. Here we describe an infant mouse model which support efficient transmission of IAV. We demonstrate that transmission in this model requires young age, close contact, shedding of virus particles from the upper respiratory tract (URT) of infected pups, the use of a transmissible virus strain, and a susceptible recipient. We characterize shedding as a marker of infectiousness that predicts the efficiency of transmission among different influenza virus strains. We also demonstrate that transmissibility and susceptibility to IAV can be inhibited by humoral immunity via maternal-infant transfer of IAV-specific immunoglobulins, and modifications to the URT milieu, via sialidase activity of colonizing Streptococcus pneumoniae (Spn). Due to its simplicity and efficiency, this model can be used to dissect the hosts contribution to IAV transmission and explore new methods to limit contagion.\n\nIMPORTANCEThis study provides insight into the role of the virus strain, age, immunity, and URT flora on IAV shedding and transmission efficiency. Using the infant mouse model, we found that: (a) differences in viral shedding of various IAV strains is dependent on specific hemagglutinin (HA) and/or neuraminidase (NA) proteins; (b) host age plays a key role in the efficiency of IAV transmission; (c) levels of IAV-specific immunoglobulins are necessary to limit infectiousness, transmission, and susceptibility to IAV; and (d) expression of sialidases by colonizing Spn antagonize transmission by limiting the acquisition of IAV in recipient hosts. Our findings highlight the need for strategies that limit IAV shedding, and the importance of understanding the function of the URT bacterial composition in IAV transmission. This work reinforces the significance of a tractable animal model to study both viral and host traits affecting IAV contagion, and its potential for optimizing vaccines and therapeutics that target disease spread.

microbiology↗