An interstrand DNA crosslink glycosylase aids Acinetobacter baumannii pathogenesis.

Maintenance of DNA integrity is essential to all forms of life. DNA damage generated by reaction with genotoxic chemicals results in deleterious mutations, genome instability, and cell death. Pathogenic bacteria encounter several genotoxic agents during infection. In keeping with this, the loss of DNA repair networks results in virulence attenuation in several bacterial species. Interstrand DNA crosslinks (ICLs) are a type of DNA lesion formed by covalent linkage of opposing DNA strands and are particularly toxic as they interfere with replication and transcription. Bacteria have evolved specialized DNA glycosylases that unhook ICLs, thereby initiating their repair. In this study, we describe AlkX, a DNA glycosylase encoded by the multidrug resistant pathogen Acinetobacter baumannii. AlkX exhibits ICL unhooking activity similar to that of its Escherichia coli homolog YcaQ. Interrogation of the in vivo role of AlkX revealed that its loss sensitizes cells to DNA crosslinking and impairs A. baumannii colonization of the lungs and dissemination to distal tissues during pneumonia. These results suggest that AlkX participates in A. baumannii pathogenesis and protects the bacterium from stress conditions encountered in vivo. Consistent with this, we found that acidic pH, an environment encountered during host colonization, results in A. baumannii DNA damage and that alkX is induced by, and contributes to, defense against acidic conditions. Collectively, these studies reveal functions for a recently described class of proteins encoded in a broad range of pathogenic bacterial species.

A multi-species outbreak of VIM-producing carbapenem-resistant bacteria in a burn unit and subsequent investigation of rapid development of cefiderocol resistance.

Carbapenem resistance due to metallo-ß-lactamases (MBLs) such as the Verona integron-encoded metallo-ß-lactamase (VIM) is particularly problematic due to the limited treatment options. We describe a case series of bacterial infections in a tertiary care hospital due to multi-species acquisition of a VIM gene along with our experience using novel ß-lactam antibiotics and antibiotic combinations to treat these infections. Four patients were treated with the combination of ceftazidime-avibactam and aztreonam, with no resistance to the combination detected. However, cefiderocol-resistant Klebsiella pneumoniae isolates were detected in two out of the five patients who received cefiderocol within 3 weeks of having started the antibiotic. Strain pairs of sequential susceptible and resistant isolates from both patients were analyzed using whole-genome sequencing. This analysis revealed that the pairs of isolates independently acquired point mutations in both the cirA and fiu genes, which encode siderophore receptors. These point mutations were remade in a laboratory strain of K. pneumoniae and resulted in a significant increase in the MIC of cefiderocol, even in the absence of a beta-lactamase enzyme or a penicillin-binding protein 3 (PBP3) mutation. While newer ß-lactam antibiotics remain an exciting addition to the antibiotic armamentarium, their use must be accompanied by diligent monitoring for the rapid development of resistance.

Restriction of arginine induces antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics are examined using both quantitative proteomics and transposon sequencing. These screens indicate that arginine metabolism is involved in antibiotic tolerance within a biofilm and support the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction induces antibiotic tolerance via inhibition of protein synthesis. In murine skin and bone infection models, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections.

FapR regulates HssRS-mediated heme homeostasis in Bacillus anthracis.

Bacillus anthracis, a Gram-positive facultative anaerobe and the causative agent of anthrax, multiplies to extraordinarily high numbers in vertebrate blood, resulting in considerable heme exposure. Heme is an essential nutrient and the preferred iron source for bacteria during vertebrate colonization, but its high redox potential makes it toxic in excess. To regulate heme homeostasis, many Gram-positive bacteria, including B. anthracis, rely on the two-component signaling system HssRS. HssRS comprises the heme sensing histidine kinase HssS, which modulates the activity of the HssR transcription factor to enable bacteria to circumvent heme toxicity. However, the regulation of the HssRS system remains unclear. Here we identify FapR, the transcriptional regulator of fatty acid biosynthesis, as a key factor in HssRS function. FapR plays an important role in maintaining membrane integrity and the localization of the histidine kinase HssS. Specifically, disruption of fapR leads to increased membrane rigidity, which hinders the penetration of HssRS inducers, resulting in the inactivation of HssRS. Furthermore, deletion of fapR affects the loading of HssS onto the cell membrane, compromising its heme sensing function and subsequently reducing endogenous heme biosynthesis. These findings shed light on the molecular mechanisms governing bacterial adaptation to heme stress and provide potential targets for antimicrobial intervention strategies.

MALDI TIMS IMS Reveals Ganglioside Molecular Diversity within Murine S. aureus Kidney Tissue Abscesses.

Gangliosides play important roles in innate and adaptive immunity. The high degree of structural heterogeneity results in significant variability in ganglioside expression patterns and greatly complicates linking structure and function. Structural characterization at the site of infection is essential in elucidating host ganglioside function in response to invading pathogens, such as Staphylococcus aureus (S. aureus). Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables high-specificity spatial investigation of intact gangliosides. Here, ganglioside structural and spatial heterogeneity within an S. aureus-infected mouse kidney abscess was characterized. Differences in spatial distributions were observed for gangliosides of different classes and those that differ in ceramide chain composition and oligosaccharide-bound sialic acid. Furthermore, integrating trapped ion mobility spectrometry (TIMS) allowed for the gas-phase separation and visualization of monosialylated ganglioside isomers that differ in sialic acid type and position. The isomers differ in spatial distributions within the host-pathogen interface, where molecular patterns revealed new molecular zones in the abscess previously unidentified by traditional histology.

Coordinated adaptation of Staphylococcus aureus to calprotectin-dependent metal sequestration.

The host protein calprotectin inhibits the growth of a variety of bacterial pathogens through metal sequestration in a process known as "nutritional immunity." Staphylococcus aureus growth is inhibited by calprotectin in vitro, and calprotectin is localized in vivo to staphylococcal abscesses during infection. However, the staphylococcal adaptations that provide defense against nutritional immunity and the role of metal-responsive regulators are not fully characterized. In this work, we define the transcriptional response of S. aureus and the role of the metal-responsive regulators, Zur, Fur, and MntR, in response to metal limitation by calprotectin exposure. Additionally, we identified genes affecting the fitness of S. aureus during metal limitation through a Transposon sequencing (Tn-seq) approach. Loss of function mutations in clpP, which encodes a proteolytic subunit of the ATP-dependent Clp protease, demonstrate reduced fitness of S. aureus to the presence of calprotectin. ClpP contributes to pathogenesis in vivo in a calprotectin-dependent manner. These studies establish a critical role for ClpP to combat metal limitation by calprotectin and reveal the genes required for S. aureus to outcompete the host for metals. IMPORTANCE: Staphylococcus aureus is a leading cause of skin and soft tissue infections, bloodstream infections, and endocarditis. Antibiotic treatment failures during S. aureus infections are increasingly prevalent, highlighting the need for novel antimicrobial agents. Metal chelator-based therapeutics have tremendous potential as antimicrobials due to the strict requirement for nutrient metals exhibited by bacterial pathogens. The high-affinity transition metal-binding properties of calprotectin represents a potential therapeutic strategy that functions through metal chelation. Our studies provide a foundation to define mechanisms by which S. aureus combats nutritional immunity and may be useful for the development of novel therapeutics to counter the ability of S. aureus to survive in a metal-limited environment.