Necrotizing fasciitis (NF) caused by flesh-eating bacteria is associated with high case fatality. In an earlier study, we reported infection of an immunocompetent individual with multiple strains of Aeromonas hydrophila (NF1-NF4), the latter three constituted a clonal group whereas NF1 was phylogenetically distinct. To understand the complex interactions of these strains in NF pathophysiology, a mouse model was used, whereby either single or mixed A. hydrophila strains were injected intramuscularly. NF2, which harbors exotoxin A (exoA) gene, was highly virulent when injected alone, but its virulence was attenuated in the presence of NF1 (exoA-minus). NF1 alone, although not lethal to animals, became highly virulent when combined with NF2, its virulence augmented by cis-exoA expression when injected alone in mice. Based on metagenomics and microbiological analyses, it was found that, in mixed infection, NF1 selectively disseminated to mouse peripheral organs, whereas the other strains (NF2, NF3, and NF4) were confined to the injection site and eventually cleared. In vitro studies showed NF2 to be more effectively phagocytized and killed by macrophages than NF1. NF1 inhibited growth of NF2 on solid media, but ExoA of NF2 augmented virulence of NF1 and the presence of NF1 facilitated clearance of NF2 from animals either by enhanced priming of host immune system or direct killing via a contact-dependent mechanism.Aeromonas hydrophila | necrotizing fasciitis | mixed infections | intramuscular mouse model | metagenomics
f Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and ⌬lpp ⌬msbB double mutants. While the ⌬ail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the ⌬lpp ⌬ail double mutant and the ⌬lpp ⌬msbB ⌬ail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD 50 ) equivalent to 6,800 LD 50 s of WT CO92. The mutant-infected animals developed balanced T H 1-and T H 2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD 50 s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the ⌬lpp ⌬msbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection. P athogenic yersiniae lead to two types of diseases: yersiniosis (typified by gastroenteritis caused by Yersinia enterocolitica and Y. pseudotuberculosis) (1) and plague (evoked by Y. pestis) (2, 3). Y. pestis has evolved from Y. pseudotuberculosis within the last 20,000 years by acquiring additional plasmids and pathogenicity islands as well as by deactivating some genes (4-6). This evolutionary adaptation allowed the plague bacterium to maintain a dual life-style in fleas and rodents/mammals and conferred the ability to survive in the blood instead of the intestine (3). Plague manifests itself in three forms: bubonic (acquired from an infected rodent through a flea bite), pneumonic (acquired either directly by aerosol transmission from an infected host's lungs through respiratory droplets or secondarily from bubonic plague), and septicemic (severe bacteremia either directly due to a flea bite or subsequent to bubonic or pneumonic plague) (2). T...
h Antibiotic resistance in medically relevant bacterial pathogens, coupled with a paucity of novel antimicrobial discoveries, represents a pressing global crisis. Traditional drug discovery is an inefficient and costly process; however, systematic screening of Food and Drug Administration (FDA)-approved therapeutics for other indications in humans offers a rapid alternative approach. In this study, we screened a library of 780 FDA-approved drugs to identify molecules that rendered RAW 264.7 murine macrophages resistant to cytotoxicity induced by the highly virulent Yersinia pestis CO92 strain. Of these compounds, we identified 94 not classified as antibiotics as being effective at preventing Y. pestis-induced cytotoxicity. A total of 17 prioritized drugs, based on efficacy in in vitro screens, were chosen for further evaluation in a murine model of pneumonic plague to delineate if in vitro efficacy could be translated in vivo. Three drugs, doxapram (DXP), amoxapine (AXPN), and trifluoperazine (TFP), increased animal survivability despite not exhibiting any direct bacteriostatic or bactericidal effect on Y. pestis and having no modulating effect on crucial Y. pestis virulence factors. These findings suggested that DXP, AXPN, and TFP may modulate host cell pathways necessary for disease pathogenesis. Finally, to further assess the broad applicability of drugs identified from in vitro screens, the therapeutic potential of TFP, the most efficacious drug in vivo, was evaluated in murine models of Salmonella enterica serovar Typhimurium and Clostridium difficile infections. In both models, TFP treatment resulted in increased survivability of infected animals. Taken together, these results demonstrate the broad applicability and potential use of nonantibiotic FDA-approved drugs to combat respiratory and gastrointestinal bacterial pathogens.
The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a highthroughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD 50 ) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD 50 when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD 50 . Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD 50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD 50 in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the ⌬lpp single or the ⌬lpp ⌬msbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD 50 . The mice infected with the ⌬lpp ⌬msbB ⌬rbsA triple mutant at 50 LD 50 were 90% protected upon subsequent challenge with 12 LD 50 of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s). Yersinia pestis is a tier 1 select agent that leads to three pathodynamic manifestations in humans, namely, bubonic, septicemic, and pneumonic plague (1). Although the disease is endemic in certain regions of the globe (2), the potential use of this organism as a biological warfare agent is a significant worldwide concern. In particular, aerosolized droplets charged with Y. pestis can lead to primary pneumonic plague and subsequent person-toperson spread, with a narrow window for antibiotic intervention (3-5). Consequently, an ideal strategy to combat the disease is to have a vaccine offering long-lasting immunity.Until 1999, a heat-killed plague vaccine composed of Y. pestis 195/P strain was availab...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.