Progressive Sub-MIC Exposure of Klebsiella pneumoniae 43816 to Cephalothin Induces the Evolution of Beta-Lactam Resistance without Acquisition of Beta-Lactamase Genes.

Bacterial exposure to antibiotic concentrations below the minimum inhibitory concentration (MIC) may result in a selection window allowing for the rapid evolution of resistance. These sub-MIC concentrations are commonly found in soils and water supplies in the greater environment. This study aimed to evaluate the adaptive genetic changes in Klebsiella pneumoniae 43816 after prolonged but increasing sub-MIC levels of the common antibiotic cephalothin over a fourteen-day period. Over the course of the experiment, antibiotic concentrations increased from 0.5 µg/mL to 7.5 µg/mL. At the end of this extended exposure, the final adapted bacterial culture exhibited clinical resistance to both cephalothin and tetracycline, altered cellular and colony morphology, and a highly mucoid phenotype. Cephalothin resistance exceeded 125 µg/mL without the acquisition of beta-lactamase genes. Whole genome sequencing identified a series of genetic changes that could be mapped over the fourteen-day exposure period to the onset of antibiotic resistance. Specifically, mutations in the rpoB subunit of RNA Polymerase, the tetR/acrR regulator, and the wcaJ sugar transferase each fix at specific timepoints in the exposure regimen where the MIC susceptibility dramatically increased. These mutations indicate that alterations in the secretion of colanic acid and attachment of colonic acid to LPS may contribute to the resistant phenotype. These data demonstrate that very low sub-MIC concentrations of antibiotics can have dramatic impacts on the bacterial evolution of resistance. Additionally, this study demonstrates that beta-lactam resistance can be achieved through sequential accumulation of specific mutations without the acquisition of a beta-lactamase gene.

Porin loss in Klebsiella pneumoniae clinical isolates impacts production of virulence factors and survival within macrophages.

Clinical isolates of Klebsiella pneumoniae are often resistant to beta-lactam antibiotics via the acquisition of extended spectrum beta lactamase (ESBL) enzymes paired with loss of one or both major outer membrane porins. It has been well established that loss of OmpK35 and/or OmpK36 correlates with increased minimum inhibitory concentrations of antibiotics that target the peptidoglycan. However, little is known concerning the downstream effects porin loss might have on other major virulence factors such as the polysaccharide capsule or LPS. Furthermore, it is unknown whether these cumulative changes impact pathogenesis. Therefore, the focus of this study was to identify alterations in production of the major virulence factors due to porin loss; and to investigate the effect these changes have on host pathogen interactions. Our data demonstrates that loss of a single porin is paired with reductions in capsule, increased LPS content, and up-regulated transcription of compensatory porin genes. In contrast, loss of both porins resulted in a significant increase in capsule production. Loss of OmpK35 alone or dual porin loss was further associated with reduced oxidative burst by macrophages and increased ability of the bacteria to survive phagocytic killing. These data indicate that porin loss is accompanied by a suite of changes in other virulence-associated factors. These cumulative changes act to nullify any negative fitness effect due to lack of the nonspecific porin proteins, allowing the bacteria to grow and survive phagocytic immune responses.

Porin Loss Impacts the Host Inflammatory Response to Outer Membrane Vesicles of Klebsiella pneumoniae.

Antibiotic-resistant strains of Klebsiella pneumoniae often exhibit porin loss. In this study, we investigated how porin loss impacted the composition of secreted outer membrane vesicles as well as their ability to trigger proinflammatory cytokine secretion by macrophages. We hypothesize that porin loss associated with antibiotic resistance will directly impact both the composition of outer membrane vesicles and their interactions with phagocytic cells. Using clonally related clinical isolates of extended-spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae with different patterns of porin expression, we demonstrated that altered expression of OmpK35 and OmpK36 results in broad alterations to the protein profile of secreted vesicles. Additionally, the level of OmpA incorporation was elevated in strains lacking a single porin. Porin loss significantly impacted macrophage inflammatory responses to purified vesicles. Outer membrane vesicles lacking both OmpK35 and OmpK36 elicited significantly lower levels of proinflammatory cytokine secretion than vesicles from strains expressing one or both porins. These data demonstrate that antibiotic resistance-associated porin loss has a broad and significant effect on both the composition of outer membrane vesicles and their interactions with phagocytic cells, which may impact bacterial survival and inflammatory reactions in the host.

Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles.

Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and cell envelope associated proteins into the environment through the production of outer membrane vesicles (OMVs). The loss of the LPS O antigen has been demonstrated in other bacterial species to significantly alter the composition of OMVs. Therefore, this study aimed to comprehensively analyze the impact of O antigen loss on the sub-proteomes of both the outer membrane and secreted OMVs from K. pneumoniae. As determined by LC-MS/MS, OMVs were highly enriched with outer membrane proteins involved in cell wall, membrane, and envelope biogenesis as compared to the source cellular outer membrane. Deletion of wbbO, the enzyme responsible for O antigen attachment to LPS, decreased but did not eliminate this enrichment effect. Additionally, loss of O antigen resulted in OMVs with increased numbers of proteins involved in post-translational modification, protein turnover, and chaperones as compared to secreted vesicles from the wild type. This alteration of OMV composition may be a compensatory mechanism to deal with envelope stress. This comprehensive analysis confirms the highly distinct protein composition of OMVs as compared to their source membrane, and provides evidence for a selective sorting mechanism that involves LPS polysaccharides. These data support the hypothesis that modifications to LPS alters both the mechanics of protein sorting and the contents of secreted OMVs and significantly impacts the protein composition of the outer membrane.

Development of a clickable activity-based protein profiling (ABPP) probe for agmatine deiminases.

Agmatine deiminases (AgDs) catalyze the hydrolytic conversion of agmatine (decarboxylated arginine) to N-carbamoylputrescine with concomitant release of ammonia. These enzymes, which are encoded by some pathogenic bacterial species, confer a competitive survival advantage by virtue of energy production and acid tolerance through agmatine catabolism. Herein we report the development of a clickable activity-based protein profiling (ABPP) probe that targets the AgD encoded by Streptococcus mutans with high selectivity and sensitivity.

Naturally produced outer membrane vesicles from Pseudomonas aeruginosa elicit a potent innate immune response via combined sensing of both lipopolysaccharide and protein components.

Pseudomonas aeruginosa is a prevalent opportunistic human pathogen that, like other Gram-negative pathogens, secretes outer membrane vesicles. Vesicles are complex entities composed of a subset of envelope lipid and protein components that have been observed to interact with and be internalized by host cells. This study characterized the inflammatory responses to naturally produced P. aeruginosa vesicles and determined the contribution of vesicle Toll-like receptor (TLR) ligands and vesicle proteins to that response. Analysis of macrophage responses to purified vesicles by real-time PCR and enzyme-linked immunosorbent assay identified proinflammatory cytokines upregulated by vesicles. Intact vesicles were shown to elicit a profoundly greater inflammatory response than the response to purified lipopolysaccharide (LPS). Both TLR ligands LPS and flagellin contributed to specific vesicle cytokine responses, whereas the CpG DNA content of vesicles did not. Neutralization of LPS sensing demonstrated that macrophage responses to the protein composition of vesicles required the adjuvantlike activity of LPS to elicit strain specific responses. Protease treatment to remove proteins from the vesicle surface resulted in decreased interleukin-6 and tumor necrosis factor alpha production, indicating that the production of these specific cytokines may be linked to macrophage recognition of vesicle proteins. Confocal microscopy of vesicle uptake by macrophages revealed that vesicle LPS allows for binding to macrophage surfaces, whereas vesicle protein content is required for internalization. These data demonstrate that macrophage sensing of both LPS and protein components of outer membrane vesicles combine to produce a bacterial strain-specific response that is distinct from those triggered by individual, purified vesicle components.

Virulence and immunomodulatory roles of bacterial outer membrane vesicles.

Outer membrane (OM) vesicles are ubiquitously produced by Gram-negative bacteria during all stages of bacterial growth. OM vesicles are naturally secreted by both pathogenic and nonpathogenic bacteria. Strong experimental evidence exists to categorize OM vesicle production as a type of Gram-negative bacterial virulence factor. A growing body of data demonstrates an association of active virulence factors and toxins with vesicles, suggesting that they play a role in pathogenesis. One of the most popular and best-studied pathogenic functions for membrane vesicles is to serve as natural vehicles for the intercellular transport of virulence factors and other materials directly into host cells. The production of OM vesicles has been identified as an independent bacterial stress response pathway that is activated when bacteria encounter environmental stress, such as what might be experienced during the colonization of host tissues. Their detection in infected human tissues reinforces this theory. Various other virulence factors are also associated with OM vesicles, including adhesins and degradative enzymes. As a result, OM vesicles are heavily laden with pathogen-associated molecular patterns (PAMPs), virulence factors, and other OM components that can impact the course of infection by having toxigenic effects or by the activation of the innate immune response. However, infected hosts can also benefit from OM vesicle production by stimulating their ability to mount an effective defense. Vesicles display antigens and can elicit potent inflammatory and immune responses. In sum, OM vesicles are likely to play a significant role in the virulence of Gram-negative bacterial pathogens.

DNA replication during aggregation phase is essential for Myxococcus xanthus development.

Previous studies have demonstrated that fruiting body-derived Myxococcus xanthus myxospores contain two fully replicated copies of its genome, implying developmental control of chromosome replication and septation. In this study, we employ DNA replication inhibitors to determine if chromosome replication is essential to development and the exact time frame in which chromosome replication occurs within the developmental cycle. Our results show that DNA replication during the aggregation phase is essential for developmental progression, implying the existence of a checkpoint that monitors chromosome integrity at the end of the aggregation phase.

Interferon-gamma activation of polymorphonuclear neutrophil function.

As current research illuminates the dynamic interplay between the innate and acquired immune responses, the interaction and communication between these two arms has yet to be fully investigated. Polymorphonuclear neutrophils (PMNs) and interferon-gamma (IFN-gamma) are known critical components of innate and acquired immunity, respectively. However, recent studies have demonstrated that these two components are not entirely isolated. Treatment of PMNs with IFN-gamma elicits a variety of responses depending on stimuli and environmental conditions. These responses include increased oxidative burst, differential gene expression, and induction of antigen presentation. Many of these functions have been overlooked in PMNs, which have long been classified as terminal phagocytic cells incapable of protein synthesis. As this review reports, the old definition of the PMN is in need of an update, as these cells have demonstrated their ability to mediate the transition between the innate and acquired immune responses.

Murine polymorphonuclear neutrophils produce interferon-gamma in response to pulmonary infection with Nocardia asteroides.

Nocardia asteroides causes an acute, necrotizing pneumonia characterized by extensive infiltration of polymorphonuclear neutrophils (PMNs) into the lungs. Although PMNs have historically been classified as end-point cells, recent investigations have indicated that PMNs have the ability to secrete cytokines such as interleukin (IL)-4 and IL-12. This study investigated the ability of PMNs to produce cytokines in a murine model of N. asteroides pulmonary infection. Flow cytometric analysis demonstrated the production of interferon-gamma (IFN-gamma), but not IL-4, by PMNs in response to this infection. IFN-gamma production correlated with peak infiltration of PMNs into the lungs. Cell sorting and enzyme-linked immunosorbent assay were used to confirm cytokine production by cells with nuclear morphology characteristic of PMNs. This is the first report of IFN-gamma production by neutrophils in response to an infection in vivo. These results suggest that PMNs play an important role in directing the host toward a T helper cell type 1 phenotypic response in the lungs.