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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Tre-DST: A Drug Susceptibility Test for Mycobacterium tuberculosis Using Solvatochromic Trehalose Probes

In 2024, an estimated 10 million people developed Tuberculosis (TB), nearly half a million of whom were infected with drug-resistant tuberculosis (DR-TB). Early detection of infection and drug resistance enables rapid engagement in effective care. Bacterial culture and nucleic acid testing remain the primary diagnostic methods, with smear microscopy being phased out. However, these methods present significant limitations for diagnosing drug resistance, such as lengthy time-to-result for phenotypic tests, as well as the need for prior knowledge of resistance mutations and prohibitive cost for molecular tests. To address this, we developed a rapid phenotypic TB drug susceptibility test, termed Tre-DST, based on novel metabolically incorporated trehalose probes, which specifically detect live mycobacteria. We used the nonpathogenic Mycobacterium smegmatis and the virulence-attenuated Mycobacterium tuberculosis (Mtb) H37Ra or auxotrophic Mtb to demonstrate a strong correlation between cost-effective plate reader results and flow cytometry data, suggesting that the plate reader is a suitable fluorescence detector for Tre-DST. We determined that adding a 1-week incubation step allowed Mtb samples originally seeded at 10 4 CFU/mL to become detectable, over 2 weeks earlier than colony-forming unit analysis. We found that Tre-DST reports on drug susceptibility in a drug-agnostic manner, demonstrating loss of fluorescence with frontline TB drugs as well as the newer drug bedaquiline. Tre-DST distinguished RIF- and INH-resistant auxotrophs from susceptible controls and accurately reported the resistance activity. Ultimately, because Tre-DST is agnostic to mechanisms of drug resistance, this assay is likely compatible with all WHO-recommended and future DR-TB drugs as a diagnostic in reference laboratories.

diagnostics↗
NASA NTRSDate not supplied

Effects of Low Dose Radiation and Radiation Countermeasures on Infection By Spaceflight Analogue Cultured Salmonella Using 3-D Biomimetic Human Tissue Models

While both microgravity and radiation are major biological stressors associated with the spaceflight environment, their cumulative impact on host-pathogen interactions and infectious disease risks are rarely considered. This is critical to address, since the cumulative effects of these stressors during spaceflight may result in unexpected negative impacts on crew health and performance that neither condition alone would predict, thus limiting the ability to develop effective countermeasures. Previously, we showed that both spaceflight and spaceflight analogue culture increased the virulence and pathogenesis-related characteristics of the foodborne pathogen, Salmonella Typhimurium (S. Typhimurium), which is responsible for disqualification of food destined for the International Space Station and Salmonella spp. have been found aboard NASA spacecraft. Recently, we demonstrated that spaceflight-analogue culture of S. Typhimurium increased its ability to infect 3-D biomimetic human intestinal tissue models. In a separate study, we showed low dose radiation damaged our 3-D intestinal models. The primary objective of this proposal is to evaluate the possibility that low dose radiation will exacerbate the already increased bacterial pathogenicity of S. Typhimurium observed following spaceflight analogue culture. In addition, we will determine the impact of a radiation countermeasure to provide protection against both radiation and pathogen-induced tissue damage and inflammation. Hypothesis: The already enhanced infection potential of spaceflight analogue cultured S. Typhimurium will be further exacerbated when used to infect host cells exposed to low dose radiation and this enhanced pathogenicity can be mitigated by a radioprotective compound. Aims: 1.Characterize the impact of spaceflight-analogue culture on the ability of S. Typhimurium to infect 3-D biomimetic intestinal tissue models before and after exposure to low dose radiation. 2. Evaluate the ability of the radioprotective compound, EC-18, to protect 3-D intestinal models from low dose radiation, S. Typhimurium infection, and the cumulative impact of these stressors. Significance: Current infectious disease risk assessments for spaceflight do not consider the potential for increased susceptibility to infection and disease resulting from exposure to low dose radiation, which is a critical consideration. This study will provide key evidence to determine if exposure to low dose radiation may be a factor in astronaut susceptibility to infection during long duration exploration missions and the impact of selected countermeasures to mitigate that risk to crew health. We have completed infection studies of the 3-D biomimetic intestinal tissue models and are currently analyzing the data.

Jennifer Barrila↗
NASA NTRS2024

Effects of Low Dose Radiation and Radiation Countermeasures on Infection By Spaceflight Analogue Cultured Salmonella Using 3-D Biometric Human Tissue Models

While both microgravity and radiation are major biological stressors associated with the spaceflight environment, their cumulative impact on host-pathogen interactions and infectious disease risks are rarely considered. This is critical to address, since the cumulative effects of these stressors during spaceflight may result in unexpected negative impacts on crew health and performance that neither condition alone would predict, thus limiting the ability to develop effective countermeasures. Previously, we showed that both spaceflight and spaceflight analogue culture increased the virulence and pathogenesis-related characteristics of the foodborne pathogen, Salmonella Typhimurium (S. Typhimurium), which is responsible for disqualification of food destined for the International Space Station and Salmonella spp. have been found aboard NASA spacecraft. Recently, we demonstrated that spaceflight-analogue culture of S. Typhimurium increased its ability to infect 3-D biomimetic human intestinal tissue models. In a separate study, we showed low dose radiation damaged our 3-D intestinal models. The primary objective of this proposal is to evaluate the possibility that low dose radiation will exacerbate the already increased bacterial pathogenicity of S. Typhimurium observed following spaceflight analogue culture. In addition, we will determine the impact of a radiation countermeasure to provide protection against both radiation and pathogen-induced tissue damage and inflammation. Hypothesis: The already enhanced infection potential of spaceflight analogue cultured S. Typhimurium will be further exacerbated when used to infect host cells exposed to low dose radiation and this enhanced pathogenicity can be mitigated by a radioprotective compound. Aims: 1. Characterize the impact of spaceflight-analogue culture on the ability of S. Typhimurium to infect 3-D biomimetic intestinal tissue models before and after exposure to low dose radiation. 2. Evaluate the ability of the radioprotective compound, EC-18, to protect 3-D intestinal models from low dose radiation, S. Typhimurium infection, and the cumulative impact of these stressors. Significance: Current infectious disease risk assessments for spaceflight do not consider the potential for increased susceptibility to infection and disease resulting from exposure to low dose radiation, which is a critical consideration. This study will provide key evidence to determine if exposure to low dose radiation may be a factor in astronaut susceptibility to infection during long duration exploration missions and the impact of selected countermeasures to mitigate that risk to crew health. This study has been initiated and data collection is beginning.

Jennifer Barrila↗
NASA NTRSDate not supplied

Effects of Low Dose Radiation and Radiation Countermeasures on Infection by Spaceflight Analogue Cultured Salmonella Using 3-D Biomimetic Human Tissue Models

While both microgravity and radiation are major biological stressors associated with the spaceflight environment, their cumulative impact on host-pathogen interactions and infectious disease risks are rarely considered. This is critical to address, since the cumulative effects of these stressors during spaceflight may result in unexpected negative impacts on crew health and performance that neither condition alone would predict, thus limiting the ability to develop effective countermeasures. Previously, we showed that both spaceflight and spaceflight analogue culture increased the virulence and pathogenesis-related characteristics of the foodborne pathogen, Salmonella Typhimurium (S. Typhimurium), which is responsible for disqualification of food destined for the International Space Station and Salmonella spp. have been found aboard NASA spacecraft. Recently, we demonstrated that spaceflight-analogue culture of S. Typhimurium increased its ability to infect 3-D biomimetic human intestinal tissue models. In a separate study, we showed low dose radiation damaged our 3-D intestinal models. The primary objective of this proposal is to evaluate the possibility that low dose radiation will exacerbate the already increased bacterial pathogenicity of S. Typhimurium observed following spaceflight analogue culture. In addition, we will determine the impact of a radiation countermeasure to provide protection against both radiation and pathogen-induced tissue damage and inflammation. Hypothesis: The already enhanced infection potential of spaceflight analogue cultured S. Typhimurium will be further exacerbated when used to infect host cells exposed to low dose radiation and this enhanced pathogenicity can be mitigated by a radioprotective compound. Aims: 1.Characterize the impact of spaceflight-analogue culture on the ability of S. Typhimurium to infect 3-D biomimetic intestinal tissue models before and after exposure to low dose radiation. 2. Evaluate the ability of the radioprotective compound, EC-18, to protect 3-D intestinal models from low dose radiation, S. Typhimurium infection, and the cumulative impact of these stressors. Significance: Current infectious disease risk assessments for spaceflight do not consider the potential for increased susceptibility to infection and disease resulting from exposure to low dose radiation, which is a critical consideration. This study will provide key evidence to determine if exposure to low dose radiation may be a factor in astronaut susceptibility to infection during long duration exploration missions and the impact of selected countermeasures to mitigate that risk to crew health. We have completed infection studies of the 3-D biomimetic intestinal tissue models and are currently analyzing the data.

Jennifer Barrila↗
NASA NTRSDate not supplied

Effects of Low Dose Radiation and Radiation Countermeasures on Infection by Spaceflight Analogue Cultured Salmonella Using 3-D Biomimetic Human Tissue Models

While both microgravity and radiation are major biological stressors associated with the spaceflight environment, their cumulative impact on host-pathogen interactions and infectious disease risks are rarely considered. This is critical to address, since the cumulative effects of these stressors during spaceflight may result in unexpected negative impacts on crew health and performance that neither condition alone would predict, thus limiting the ability to develop effective countermeasures. Previously, we showed that both spaceflight and spaceflight analogue culture increased the virulence and pathogenesis-related characteristics of the foodborne pathogen, Salmonella Typhimurium (S. Typhimurium), which is responsible for disqualification of food destined for the International Space Station and Salmonella spp. have been found aboard NASA spacecraft. Recently, we demonstrated that spaceflight-analogue culture of S. Typhimurium increased its ability to infect 3-D biomimetic human intestinal tissue models. In a separate study, we showed low dose radiation damaged our 3-D intestinal models. The primary objective of this proposal is to evaluate the possibility that low dose radiation will exacerbate the already increased bacterial pathogenicity of S. Typhimurium observed following spaceflight analogue culture. In addition, we will determine the impact of a radiation countermeasure to provide protection against both radiation and pathogen-induced tissue damage and inflammation. Hypothesis: The already enhanced infection potential of spaceflight analogue cultured S. Typhimurium will be further exacerbated when used to infect host cells exposed to low dose radiation and this enhanced pathogenicity can be mitigated by a radioprotective compound. Aims: 1. Characterize the impact of spaceflight-analogue culture on the ability of S. Typhimurium to infect 3-D biomimetic intestinal tissue models before and after exposure to low dose radiation. 2. Evaluate the ability of the radioprotective compound, EC-18, to protect 3-D intestinal models from low dose radiation, S. Typhimurium infection, and the cumulative impact of these stressors. Significance: Current infectious disease risk assessments for spaceflight do not consider the potential for increased susceptibility to infection and disease resulting from exposure to low dose radiation, which is a critical consideration. This study will provide key evidence to determine if exposure to low dose radiation may be a factor in astronaut susceptibility to infection during long duration exploration missions and the impact of selected countermeasures to mitigate that risk to crew health. This study has been initiated and data collection is beginning.

Jennifer Barrila↗
DOE OSTI2025

Bleach Rescues Nannochloropsis from an Obligate Parasite and Alters Microbial and Metabolite Signatures of Outdoor Cultures

Chemical agents are commonly used to protect algal crops. Yet, few studies have characterized the effects of these agents on associated microbial communities to understand effects on microbial functions relevant to algal crop production and protection. Here, we used shotgun metagenomic sequencing and untargeted exometabolite profiling to link the application of bleach, a -cidal agent used to protect algae from pests, to changes in community composition, metabolic pathways, and exometabolies - at a whole community level. Bleach protected the algal crop from crashing but altered bacterial diversity. Analysis of metagenome-assembled genomes (MAGs) revealed a classic predator-prey cycle between Oligoflexus and our target alga Nannochloropsis. Olifoflexus genomes from our study were notably similar to a previously identified BALO (Bdellovibrio and like organism), FD111, known to kill Nannochloropsis cultures, providing strong evidence that an FD111-like organism was responsible for the crash. Metabolic pathway composition differed between bleached and unbleached ponds, with abundance of twelve pathways related to stress tolerance, including the superpathway of methylglyoxal degradation, lipid IVA biosynthesis, and ectoine biosynthesis, greater in bleached ponds compared to unbleached ponds. Virulence factors related to adherence, biofilm formation, motility, and pathogenicity increased dramatically in bleached ponds with time, although this increase was not coupled with an increase in pathogens - algal or otherwise - or a decline in algal health. Our study highlights the importance of coupling 16S rRNA gene sequencing with whole genome data and other -omics tools to sketch a larger picture of community structure and function in crop systems. Moreover, our results highlight that continued long-term bleaching may lead to negative effects to crop health or downstream adverse health effects to humans or animals, depending on the algal product (i.e. human supplements or animal feedstocks). Future work on alternative treatment methods that would reduce resistance is necessary in the field.

09 BIOMASS FUELS↗
DOE OSTI2021

Structural basis for antibody binding to adenylate cyclase toxin reveals RTX linkers as neutralization-sensitive epitopes

RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type 1 secretion system (T1SS) and target leukocytes to subvert host defenses. T1SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T1SS and drives secretion via a Brownian ratchet mechanism. Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin, an RTX leukotoxin essential for B . pertussis colonization, have been shown to target the RTX domain and prevent binding to the α M β 2 integrin receptor. Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform structure-based design of bacterial vaccines, however, no structural data are available for antibody binding to any T1SS substrate. Here, we determine the crystal structure of an engineered RTX domain fragment containing the α M β 2 -binding site bound to two neutralizing antibodies. Notably, the receptor-blocking antibodies bind to the linker regions of RTX blocks I–III, suggesting they are key neutralization-sensitive sites within the RTX domain and are likely involved in binding the α M β 2 receptor. As the engineered RTX fragment contained these key epitopes, we assessed its immunogenicity in mice and showed that it elicits similar neutralizing antibody titers to the full RTX domain. The results from these studies will support the development of bacterial vaccines targeting RTX leukotoxins, as well as next-generation B . pertussis vaccines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Identification of a secretory heme‐binding protein from Nocardia seriolae involved in cell apoptosis

Abstract According to the whole‐genome bioinformatics analysis, a heme‐binding protein from Nocardia seriolae (HBP) was found. HBP was predicted to be a bacterial secretory protein, located at mitochondrial membrane in eukaryotic cells and have a similar protein structure with the heme‐binding protein of Mycobacterium tuberculosis , Rv0203. In this study, HBP was found to be a secretory protein and co‐localized with mitochondria in FHM cells. Quantitative analysis of mitochondrial membrane potential value, caspase‐3 activity, and transcription level of apoptosis‐related genes suggested that overexpression of HBP protein can induce cell apoptosis. In conclusion, HBP was a secretory protein which may target to mitochondria and involve in cell apoptosis in host cells. This research will promote the function study of HBP and deepen the comprehension of the virulence factors and pathogenic mechanisms of N. seriolae .

Wen, Yiming↗
NASA NTRS2017

Effects of Simulated Microgravity on a Host-Pathogen System

While it has been shown that decades of astronauts and cosmonauts can suffer from illnesses both during and after spaceflight, the underlying causes are still poorly understood, due in part to the fact that there are so many variables to consider when investigating the human immune system in a complex environment. Invertebrates have become popular models for studying human disease because they are cheap, highly amenable to experimental manipulation, and have innate immune systems with a high genetic similarity to humans. Fruit flies (Drosophila melanogaster) have been shown to experience a dramatic shift in immune gene expression following spaceflight, but are still able to fight off infections when exposed to bacteria. However, the common bacterial pathogen Serratia marcescens was shown to become more lethal to fruit flies after being cultured in space, suggesting that not only do we need to consider host changes in susceptibility, but also changes in the pathogen itself after spaceflight conditions. Being able to simulate spaceflight conditions in a controlled environment on the ground gives us the ability to not only evaluate the effects of microgravity on the host immune system, but also how the microorganisms that cause immune disorders are being affected by these drastic environmental shifts. In this study, I use a ground-based simulated microgravity environment to examine the genetic changes associated with increased S. marcescens virulence in order to understand how microgravity is affecting this pathogen, as well as how these genetic changes influence and interact with the host immune system. This study will provide us with more directed approaches to studying the effects of spaceflight on human beings, with the ultimate goal of being able to counteract immune dysfunction in future space exploration.

Immunology↗
NASA NTRS2017

Studying the Effect of Radiation in the Context of Deep Space Travel

While it has been shown that decades of astronauts and cosmonauts can suffer from illnesses both during and after spaceflight, the underlying causes are still poorly understood, due in part to the fact that there are so many variables to consider when investigating the human immune system in a complex environment. Invertebrates have become popular models for studying human disease because they are cheap, highly amenable to experimental manipulation, and have innate immune systems with a high genetic similarity to humans. Fruit flies (Drosophila melanogaster) have been shown to experience a dramatic shift in immune gene expression following spaceflight, but are still able to fight off infections when exposed to bacteria. However, the common bacterial pathogen Serratia marcescens was shown to become more lethal to fruit flies after being cultured in space, suggesting that not only do we need to consider host changes in susceptibility, but also changes in the pathogen itself after spaceflight conditions. Being able to simulate spaceflight conditions in a controlled environment on the ground gives us the ability to not only evaluate the effects of microgravity on the host immune system, but also how the microorganisms that cause immune disorders are being affected by these drastic environmental shifts. In this study, I use a ground-based simulated microgravity environment to examine the genetic changes associated with increased S. marcescens virulence in order to understand how microgravity is affecting this pathogen, as well as how these genetic changes influence and interact with the host immune system. This study will provide us with more directed approaches to studying the effects of spaceflight on human beings, with the ultimate goal of being able to counteract immune dysfunction in future space exploration.

Bhattacharya, Sharmila↗
NASA NTRS2022

Asparagine Biosynthesis as a Mechanism of Increased Host Lethality Induced by Serratia Marcescens in Simulated Microgravity Environments

While studies have shown an increase in pathogenicity in several microbes during spaceflight and after exposure to simulated microgravity, the mechanisms underlying these changes in phenotype are not understood across different pathogens, particularly in opportunistic pathogens. This study evaluates the mechanism for increased virulence of the opportunistic gram-negative bacterium, Serratia marcescens, in simulated microgravity. Low-shear modeled microgravity (LSMMG) is used in ground-based studies to simulate the effects of microgravity as experienced in spaceflight. Our previous findings showed that there was a significant increase in mortality rates of the Drosophila melanogaster host when infected with either spaceflight or LSMMG treated S. marcescens. Here, we report that LSMMG increases asparagine uptake and synthesis in S. marcescens and that the increased host lethality induced by LSMMG bacteria grown in rich media can be recapitulated in minimal media by adding only aspartate and glutamine, the substrates of asparagine biosynthesis. Interestingly, increased bacterial growth rate alone is not sufficient to contribute to maximal host lethality, since the addition of aspartate to minimal media caused an LSMMG-specific increase in bacterial growth rate that is comparable to that induced by the combination of aspartate and glutamine, but this increase in growth does not cause an equivalent rate of host mortality. However, the addition of both aspartate and glutamine cause both an increase in host mortality and an overexpression of asparagine pathway genes in a LSMMG-dependent manner. We also report that L-asparaginase-mediated breakdown of asparagine is an effective countermeasure for the increased host mortality caused by LSMMG-treated bacteria. This investigation underscores the importance of the asparagine utilization pathway by helping uncover molecular mechanisms that underlie increased mortality rates of a model host infected with microgravity-treated S.marcescens and provides a potential mitigation strategy.

simulated microgravity↗
DOE OSTI2025

Infection and Genomic Properties of Single- and Double-Stranded DNA Cellulophaga Phages

Bacterial viruses (phages) are abundant and ecologically impactful, but laboratory-based experimental model systems vastly under-represent known phage diversity, particularly for ssDNA phages. Here, we characterize the genomes and infection properties of two unrelated marine flavophages—ssDNA generalist phage phi18:4 (6.5 Kbp) and dsDNA specialist phage phi18:1 (39.2 Kbp)—when infecting the same Cellulophaga baltica strain #18 (Cba18), of the class Flavobacteriia. Phage phi18:4 belongs to a new family of ssDNA phages, has an internal lipid membrane, and its genome encodes primarily structural proteins, as well as a DNA replication protein common to ssDNA phages and a unique lysis protein. Phage phi18:1 is a siphovirus that encodes several virulence genes, despite not having a known temperate lifestyle, a CAZy enzyme likely for regulatory purposes, and four DNA methyltransferases dispersed throughout the genome that suggest both host modulation and phage DNA protection against host restriction. Physiologically, ssDNA phage phi18:4 has a shorter latent period and smaller burst size than dsDNA phage phi18:1, and both phages efficiently infect this host. These results help augment the diversity of characterized environmental phage–host model systems by studying infections of genomically diverse phages (ssDNA vs. dsDNA) on the same host.

Howard-Varona, Cristina (ORCID:0000000241495818)↗
NASA NTRS2018

Effects of Simulated Microgravity on a Host-Pathogen System

While it has been shown that decades of astronauts and cosmonauts suffer from immune disorders both during and after spaceflight, the underlying causes are still poorly understood, due in part to the fact that there are so many variables to consider when investigating the human immune system in a complex environment. Invertebrates have become popular models for studying human disease because they are cheap, highly amenable to experimental manipulation, and have innate immune systems with a high genetic similarity to humans. Fruit flies (Drosophila melanogaster) have been shown to experience a dramatic shift in immune gene expression following spaceflight, but are still able to fight off infections when exposed to bacteria. Furthermore, a recent spaceflight mission showed that flies are more susceptible to infection following exposure to microgravity conditions, compared to ground-reared flies from the same population. Additionally, the common bacterial pathogen Serratia marcescens was shown to become more lethal to fruit flies (both space- and ground-reared) after being cultured in space, suggesting that not only do we need to consider host changes in susceptibility, but also changes in the pathogen itself after spaceflight conditions. Being able to simulate spaceflight conditions in a controlled environment on the ground gives us the ability to not only evaluate the effects of microgravity on the host immune system, but also how the microorganisms that cause immune disorders are being affected by these drastic environmental shifts. In this study, I use both spaceflight and ground-based (simulated microgravity) environments to examine the genetic changes associated with increased S. marcescens virulence in order to understand how microgravity is affecting this pathogen, as well as to evaluate how these genetic changes influence and interact with the host immune system. This study will provide us with more directed approaches to studying the effects of spaceflight on human beings, with the ultimate goal of being able to ameliorate human immune dysfunction in future space exploration.

Immunology↗
NASA NTRS1993

Phenotypic characterization of xpr, a global regulator of extracellular virulence factors in Staphylococcus aureus

We recently described a Tn551 insertion in the chromosome of Staphylococcus aureus S6C that resulted in drastically reduced expression of extracellular lipase (M. S. Smeltzer, S. R. Gill, and J. J. Iandolo, J. Bacteriol. 174:4000-4006, 1992). The insertion was localized to a chromosomal site (designated omega 1058) distinct from the lipase structural gene (geh) and the accessory gene regulator (agr), both of which were structurally intact in the lipase-negative (Lip-) mutants. In this report, we describe a phenotypic comparison between strains S6C, a hyperproducer of enterotoxin B; KSI9051, a derivative of S6C carrying the Tn551 insertion at omega 1058; ISP546, an 8325-4 strain that carries a Tn551 insertion in the agr locus; and ISP479C, the parent strain of ISP546 cured of the Tn551 delivery plasmid pI258repA36. Compared with their respective parent strains, ISP546 and KSI9051 produced greatly reduced amounts of lipase, alpha-toxin, delta-toxin, protease, and nuclease. KSI9051 also produced reduced amounts of staphylococcal enterotoxin B. Coagulase production was increased in ISP546 but not in KSI9051. Using a mouse model, we also demonstrated that ISP546 and KSI9051 were far less virulent than ISP479C and S6C. We have designated the genetic element defined by the Tn551 insertion at omega 1058 xpr to denote its role as a regulator of extracellular protein synthesis. We conclude that xpr and agr are similar and possibly interactive regulatory genes that play an important role in pathogenesis of staphylococcal disease.

NASA Discipline Cell Biology↗
DOE OSTI2024

The multifaceted role of c-di-AMP signaling in the regulation of Porphyromonas gingivalis lipopolysaccharide structure and function

This study unveils the intricate functional association between cyclic di-3’,5’-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLAT n . We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Salmonella Typhoid Toxin PltB Subunit and Its Non-typhoidal Salmonella Ortholog Confer Differential Host Adaptation and Virulence

Typhoidal and non-typhoidal Salmonelleae (NTS) cause typhoid fever and gastroenteritis, respectively, in humans. Salmonella typhoid toxin contributes to typhoid disease progression and chronic infection, but little is known about the role of its NTS ortholog. We found that typhoid toxin and its NTS ortholog induce different clinical presentations. The PltB subunit of each toxin exhibits different glycan-binding preferences that correlate with glycan expression profiles of host cells targeted by each bacterium at the primary infection or intoxication sites. Through co-crystal structures of PltB subunits bound to specific glycan receptor moieties, we show that they induce markedly different glycan-binding preferences and virulence outcomes. Furthermore, immunization with the NTS S. Javiana or its toxin offers cross-reactive protection against lethal-dose typhoid toxin challenge. Cumulatively, these results offer insights into the evolution of host adaptations in Salmonella AB toxins, their cell and tissue tropisms, and the design for improved typhoid vaccines and therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Genomic reconstruction of Bacillus anthracis from complex environmental samples enables high-throughput identification and lineage assignment in Pakistan

Bacillus anthracis, the causative agent of anthrax, is a highly virulent zoonotic pathogen primarily affecting domesticated and wild herbivores. Human exposure to B. anthracis is primarily through contact with infected animals or contaminated animal products. In Pakistan, where livestock vaccines are largely unavailable and infected carcasses are often disposed of improperly, the risk to humans, wildlife and livestock is significant. Currently, the diagnosis of anthrax infections and outbreak tracing necessitates the isolation and culturing of B. anthracis, a process that requires BSL-3 facilities. In this study, we show that positive identification, genome reconstruction and lineage assignment can be accomplished using bioinformatic analysis of DNA extracted directly from environmental samples that would otherwise provide the starting material for isolation and culturing. This approach does not require laboratory target enrichment as is necessary for other pathogens, due in part to the extremely high bacterial load in the bloodstream in the deceased animals. Using these methods, we greatly expand the knowledge of endemic B. anthracis in Pakistan. We provide the first reference B. anthracis genomes from Pakistan since the 1970s and identify A.Br.014 Aust94 as a minor circulating sublineage alongside the dominant A.Br.047 Vollum. Future work will focus on the limits of detection and will determine if this bioinformatic method can be expanded more broadly for B. anthracis or other pathogens to replace typical culture-based methods.

A.Br.047 Vollum↗
DOE OSTI2021

The Enzymatic Activity of Inosine 5'-Monophosphate Dehydrogenase May Not Be a Vulnerable Target for Staphylococcus aureus Infections

Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 μM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. In this work, five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

60 APPLIED LIFE SCIENCES↗