Biology subjects

Mike, L. A.

Publications and source records attributed to Mike, L. A..

9 recordsLinked to original sources

bioRxiv2026

Lipopolysaccharide and Wzi retain capsular polysaccharide at the Klebsiella pneumoniae cell surface and promote bloodstream infection fitness

Bacterial capsules are formed by individual capsular polysaccharide (CPS) polymers and are the outermost extracellular layers of many Gram-positive and Gram-negative bacteria. Capsules affect several aspects of bacterial biology, including resistance to host immune factors, survival in unfavorable environments and antimicrobial resistance. Research on CPS biology has largely focused on its synthesis and the pathogenic consequences of its presence or absence. Beyond synthesis, our understanding of its retention (attachment) on the cell surface is limited. Klebsiella pneumoniae is an important Gram-negative pathogen and expresses more than 80 capsular serotypes using the common Wzy-dependent CPS biosynthesis system and phenotypically manifests in various forms, making this species an attractive bacterial model to study the underexplored aspects of CPS biology. Here, we identified and characterized bacterial components involved in CPS retention in K. pneumoniae and assessed the importance of CPS retention on bloodstream fitness. Our examination revealed that lipopolysaccharide (LPS) and the outer-membrane protein, Wzi, collectively retain K. pneumoniae CPS on the bacterial surface. We demonstrate that the core-oligosaccharide domain of LPS and the extracellular surface of Wzi likely provide anchoring sites for CPS on the bacterial surface. Furthermore, CPS retention depends on divalent cation availability. Importantly, CPS retention on the bacterial surface is important for survival in serum and bacterial fitness at secondary infection sites in a murine bacteremia model. Together, our findings demonstrate that the functional importance of CPS to bacteria extends beyond synthesis and its overall abundance. Bacterial control over other CPS properties, like cell surface retention, are also important for K. pneumoniae fitness during bacteremia.

microbiology↗

bioRxiv2026

Phenotypic heterogeneity and kidney tropism of Klebsiella pneumoniae clinical urinary tract infection isolates

Urinary tract infections (UTIs) are a significant public health burden that impact millions of people every year and are highly prevalent among in hospital-acquired infections. Klebsiella pneumoniae is the second most common cause of UTIs after uropathogenic Escherichia coli (UPEC). Thus far, the molecular mechanisms underlying pathogenesis is better understood in UPEC than K. pneumoniae. UPEC is known to have fitness factors such as fimbrial adhesion and evasion of complement-mediated killing. In other infection types, K. pneumoniae fitness has been associated with mucoidy and diverse capsular serotypes. To establish K. pneumoniae virulence factors contributing to UTI, we examined how environmental cues regulate urovirulence-associated phenotypes in clinical K. pneumoniae UTI strains. These factors included capsular polysaccharide properties, hemagglutination, serum resistance, adherence to bladder epithelial cells, and in vivo fitness. We found that clinical K. pneumoniae UTI isolates phenotypes are highly heterogeneous and can change in response to human urine. Despite K. pneumoniae clinical isolates presenting heterogeneous fitness properties, all similarly colonize the urinary tract. These results suggest that additional fitness factors contribute to K. pneumoniae uropathogenesis. Identifying these shared fitness factors will provide mechanistic insights into Klebsiella uropathogenesis and reveal candidate therapeutic targets.

microbiology↗

bioRxiv2025

Sugar Import Suppresses Klebsiella pneumoniae Mucoidy in cAMP-CRP-dependent Manner

Klebsiella pneumoniae causes over 700,000 global deaths annually and this number continues to rise due to increasing antimicrobial resistance. Hypervirulent K. pneumoniae (hvKp) often causes severe invasive infections in community and hospital settings, typically originating from gut colonization. Capsular polysaccharide (CPS) and the associated features are a key hvKp virulence factor. Altering CPS properties, such as mucoidy, in response to environmental cues enhances K. pneumoniae fitness. Although several physical and nutrient cues influence mucoidy, the molecular mechanisms by which host-relevant signals, such as sugars, regulate this key CPS property remain undefined. Here, we show that sugar import, not catabolism, broadly suppresses hvKp mucoidy via cAMP-CRP signaling. Sugars suppress mucoidy by decreasing rmpADC promoter activity and the expression of the mucoidy regulator, rmpD. Moreover, this sugar-dependent regulation is conserved across multiple hvKp strains. hvKp associates with gut mucin and gut epithelial cells at a greater frequency when in a non-mucoid state. Together, our results suggest that sugar import broadly suppresses hvKp mucoidy and increases bacterial association to host cells.

microbiology↗

bioRxiv2025

Connections between Klebsiella pneumoniae Bloodstream Dynamics and Serotype-Independent Capsule Properties

Klebsiella pneumoniae bacteremia is a significant public health burden with a 26% mortality rate, which increases when the infecting isolate is multi-drug resistant. An important virulence factor of K. pneumoniae is the capsule, the protective polysaccharide coat which surrounds the outer membrane and is made up of individual capsular polysaccharide (CPS) chains. Capsule can differ in abundance, attachment, and length of the individual CPS chains. Long, uniform CPS chains are associated with a high level of mucoidy. Typically, mucoid CPS is produced by the hypervirulent K. pneumoniae (hvKp) pathotype, which is associated with invasive community-acquired infections. In contrast, the classical K. pneumoniae (cKp) pathotype tends to be non-mucoid and is associated with nosocomial infections and multi-drug resistance. There are over 80 serotypes of K. pneumoniae capsule. Capsule swap experiments have begun to reveal the effect of serotype on virulence and immune interactions. Clinically, the K2 capsule serotype is a common serotype associated with neonatal sepsis cases. Both cKp and hvKp can produce K2 capsule, but how K2-encoding cKp and hvKp strains differ in a bloodstream infection remains unknown. To fill this gap in knowledge, we characterized the surface properties of K2 serotype cKp and hvKp bloodstream infection isolates, then tested the fitness of these strains in bloodstream infection-related in vitro and in vivo assays. Understanding how K2 cKp and hvKp strains differ in pathogenic potential provides further insight into how K. pneumoniae capsule properties influence bloodstream infection pathogenesis. IMPORTANCEMany studies have compared the pathogenesis of the two Klebsiella pneumoniae pathotypes, but did not control for differences in capsule serotype. In this study, we control for the K2 capsule serotype in classical and hypervirulent pathotypes. Our studies reveal that despite the two pathotypes exhibiting similar human serum survival, they display different tissue tropism in a murine bloodstream infection model. Additionally, although these pathotypes produce similar amounts of capsule, the hypervirulent strains are significantly more mucoid. Our study has provided more evidence on how capsule characteristics impact K. pneumoniae bacteremia pathogenesis and the importance of capsule serotype.

microbiology↗

bioRxiv2024

Arginine Regulates the Mucoid Phenotype of Hypervirulent Klebsiella pneumoniae

Hypervirulent Klebsiella pneumoniae is associated with severe community-acquired infections. Hypervirulent K. pneumoniae colonies typically exhibit a mucoid phenotype. K. pneumoniae mucoidy is influenced by a complex combination of environmental factors and genetic mechanisms. Mucoidy results from altered capsular polysaccharide chain length, yet the specific environmental cues regulating this phenotype and their impact on pathogenesis remain unclear. This study demonstrates that casamino acids enhance the mucoidy phenotype but do not affect total capsular polysaccharide levels. Through targeted screening of each amino acid present in casamino acids, we identified that arginine is necessary and sufficient to stimulate the mucoid phenotype without altering capsule abundance. Furthermore, arginine activates the rmpADC promoter, increasing rmpD transcript levels, which in turn modulates capsular polysaccharide chain length and diversity. The arginine regulator, ArgR, plays a pivotal role in this regulatory cascade since deleting argR decreases mucoidy and increases capsular polysaccharide chain length diversity. Additionally, the {Delta}argR mutant displays increased macrophage association and has a substantial competitive defect in the lungs of mice, suggesting a link between arginine-dependent gene regulation, immune evasion and in vivo fitness. We discovered that arginine-dependent regulation of mucoidy is conserved in four additional hypervirulent K. pneumoniae isolates likely via a conserved ARG binding box present in rmp promoters. Our findings support a model in which arginine activates ArgR and increases mucoidy in hypervirulent K. pneumoniae. As a result, it is possible that arginine-dependent regulation of mucoidy allows hypervirulent K. pneumoniae to adapt the cell surface across different niches. This study underscores the significance of arginine as a regulatory signal in bacterial virulence. IMPORTANCEThe rise of hypervirulent Klebsiella pneumoniae as a global health threat underscores the urgent need to understand its pathogenic mechanisms. Its ability to cause severe infections in healthy individuals and spread beyond the endemic Asia-Pacific region demands a deeper investigation into the mechanisms driving hypervirulence. The hypermucoid phenotype is primarily associated with hypervirulent isolates and is regulated by RmpD, which increases capsular polysaccharide chain length and uniformity. Understanding how environmental and genetic factors influence mucoidy is vital for elucidating the mechanisms by which K. pneumoniae adapts and thrives in different ecological and host niches. Our study defines the role of amino acids, particularly arginine, in regulating the bacterial surface by modulating rmpD expression. These results reveal that mucoidy is not a constitutive phenotype, but rather a dynamic process finely tuned by nutrient availability. Our findings expand our understanding of how the rmp locus is controlled and how changes in arginine availability may optimize K. pneumoniae immune evasion.

microbiology↗

bioRxiv2022

The Klebsiella pneumoniae ter operon enhances stress tolerance

Healthcare-acquired infections are a leading cause of disease in patients that are hospitalized or in long-term care facilities. Klebsiella pneumoniae (Kp) is a leading cause of bacteremia, pneumonia, and urinary tract infections in these settings. Previous studies have established that the ter operon, a genetic locus that confers tellurite oxide (K2TeO3) resistance, is associated with infection in colonized patients. Rather than enhancing fitness during infection, the ter operon increases Kp fitness during gut colonization; however, the biologically relevant function of this operon is unknown. First, using a murine model of urinary tract infection, we demonstrate a novel role for the ter operon protein TerC as a bladder fitness factor. To further characterize TerC, we explored a variety of functions, including resistance to metal-induced stress, resistance to ROS-induced stress, and growth on specific sugars, all of which were independent of TerC. Then, using well-defined experimental guidelines, we determined that TerC is necessary for tolerance to ofloxacin, polymyxin B, and cetylpyridinium chloride. We used an ordered transposon library constructed in a Kp strain lacking the ter operon to identify genes required to resist K2TeO3- and polymyxin B-induced stress, which suggested that K2TeO3-induced stress is experienced at the bacterial cell envelope. Finally, we confirmed that K2TeO3 disrupts the Kp cell envelope, though these effects are independent of ter. Collectively, the results from these studies indicate a novel role for the ter operon as stress tolerance factor, therefore explaining its role in enhancing fitness in the gut and bladder.

microbiology↗

bioRxiv2022

Regulation of Klebsiella pneumoniae mucoidy by the bacterial tyrosine kinase Wzc

Klebsiella pneumoniae is a frequent nosocomial pathogen associated with urinary tract infections (UTI), pneumonia, and septicemia. Two lineages of K. pneumoniae have emerged that challenge the treatment of these infections. One lineage includes hypervirulent strains, causing invasive community-acquired infections, and the other encompasses carbapenem-resistant isolates. Non-mucoid, carbapenem-resistant isolates are most frequently isolated from UTI. Therefore, we hypothesized that environmental conditions may drive K. pneumoniae adaptation to the urinary tract. We found that two rmp-positive, hypervirulent strains and two rmp-negative clinical UTI isolates all suppressed mucoidy when cultured in urine compared to rich LB medium without altering capsular polysaccharide (CPS) abundance. We then performed a transposon screen to identify genetic factors through which urine suppresses mucoidy. Due to variations within transposon hits, we performed whole genome sequencing on our hits and found 100% association between secondary mutations in Wzc and increased mucoidy. Wzc is an inner membrane tyrosine kinase that regulates CPS assembly. When Wzc mutations are encoded chromosomally we observed both increased mucoidy and increased cell-free extracellular polysaccharide (EPS). However, when Wzc mutations are expressed in trans we observed increased mucoidy without impacting CPS or cell-free EPS abundance. Functionally, one periplasmic mutation increased Wzc autophosphorylation, while four active site mutations reduced Wzc autophosphorylation. These four active site mutations are also sufficient to increase mucoidy in one of the rmp-negative clinical UTI isolates without affecting EPS abundance. Combined, these data implicate mutation-driven modulation of Wzc activity as a global, rmp-independent mechanism for increasing K. pneumoniae mucoidy, without altering EPS uronic acid content. Wzc activity may represent the lynchpin that coordinates both capsule biosynthesis and mucoidy, explaining why these two phenotypes have been historically intertwined.

microbiology↗

bioRxiv2020

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness

Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been stymied by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying levels of CPS production and hmv, were all significantly out-competed by wildtype in a murine model of disseminating pneumonia. This suggests that an optimal balance between cellular energetics, CPS biosynthesis and hmv are required for maximal fitness. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that these processes are integrated into the metabolic status of the cell. Therefore, hvKp may require certain nutrients to fully elaborate its virulence-associated properties to specifically cause deep tissue infections. Author summaryKlebsiella pneumoniae is a common multi-drug resistant hospital-associated pathogen, however some isolates are capable of causing community-acquired infections in otherwise healthy individuals. The strains causing community-acquired infections have some distinguishing characteristics, which include overproduction of capsule and hypermucoviscosity. Hypermucoviscous strains are very tacky and sediment poorly when centrifuged. Historically, hypermucoviscosity has been attributed to overproduction of capsular polysaccharide, but recent data suggest that other factors contribute to this bacterial phenotype. Moreover, it seems that capsule and hypermucoviscosity may have distinct roles in pathogenesis. In this study, we sought to systematically investigate the genes that contribute to capsule and hypermucoviscosity. We found that in most cases, genes coordinately impact both capsule biosynthesis and hypermucoviscosity. Some metabolic genes linked to the TCA cycle, however, only affect one of these properties. Here, we identify that capsule biosynthesis and hypermucoviscosity are tightly tied to central metabolism and that an optimal balance between metabolism, capsule, and hypermucoviscosity are important for in vivo fitness of K. pneumoniae. These results identify genes that can be further probed to dissect how capsule and hypermucoviscosity are coordinated in response to niche-specific nutrients. Such studies will expand our understanding of the factors that drive the pathobiology of hypervirulent K. pneumoniae.

microbiology↗

bioRxiv2020

Optimization of an Experimental Vaccine to Prevent Escherichia coli Urinary Tract Infection

Urinary tract infections (UTI) affect half of all women at least once during their lifetime. The rise in extended-spectrum beta-lactamase-producing strains and potential for carbapenem resistance within uropathogenic Escherichia coli (UPEC), the most common causative agent of UTIs, creates an urgent need for vaccine development. Intranasal immunization of mice with UPEC outer membrane iron receptors, FyuA, Hma, IreA, or IutA, conjugated to cholera toxin, provides protection in the bladder or kidneys when challenged with UPEC CFT073 or 536. Based on these data, we sought to optimize the vaccination route (intramuscular, intranasal, or subcutaneous) in combination with adjuvants suitable for human use including alum, monophosphoryl lipid A (MPLA), unmethylated CpG synthetic oligodeoxynucleotides (CpG), polyinosinic:polycytodylic acid (polyIC), and mutated heat-labile E. coli enterotoxin (dmLT). Mice intranasally vaccinated with dmLT-IutA or dmLT-Hma displayed a significant reduction in bladder colonization (86-fold and 32-fold, respectively) with 40-42% of mice having no detectable colony forming units (CFU). Intranasal vaccination of mice with CpG-IutA and polyIC-IutA significantly reduced kidney colonization (131-fold) and urine CFU (22-fold), respectively. dmLT generated the most consistently robust antibody response in intranasally immunized mice, while MPLA and alum produced greater concentrations of antigen-specific serum IgG with intramuscular immunization. Based on these results, we conclude that intranasal administration of Hma or IutA formulated with dmLT adjuvant provides the greatest protection from UPEC UTI. This study advances our progress toward a vaccine against uncomplicated UTI, which will significantly improve the quality of life for women burdened by recurrent UTI and enable better antibiotic stewardship. ImportanceUrinary tract infections (UTI) are among the most common bacterial infection in humans, affecting half of all women at least once during their lifetimes. The rise in antibiotic resistance and health care costs emphasizes the need to develop a vaccine against the most common UTI pathogen, Escherichia coli. Vaccinating mice intranasally with a detoxified heat-labile enterotoxin and two surface exposed receptors, Hma or IutA, significantly reduced bacterial burden in the bladder. This work highlights progress in the development of a UTI vaccine formulated with adjuvants suitable for human use and antigens that encode outer membrane iron receptors required for infection in the iron-limited urinary tract.

microbiology↗