Genetic requirements for Staphylococcus aureus nitric oxide resistance and virulence.

Staphylococcus aureus exhibits many defenses against host innate immunity, including the ability to replicate in the presence of nitric oxide (NO·). S. aureus NO· resistance is a complex trait and hinges on the ability of this pathogen to metabolically adapt to the presence of NO·. Here, we employed deep sequencing of transposon junctions (Tn-Seq) in a library generated in USA300 LAC to define the complete set of genes required for S. aureus NO· resistance. We compared the list of NO·-resistance genes to the set of genes required for LAC to persist within murine skin infections (SSTIs). In total, we identified 168 genes that were essential for full NO· resistance, of which 49 were also required for S. aureus to persist within SSTIs. Many of these NO·-resistance genes were previously demonstrated to be required for growth in the presence of this immune radical. However, newly defined genes, including those encoding SodA, MntABC, RpoZ, proteins involved with Fe-S-cluster repair/homeostasis, UvrABC, thioredoxin-like proteins and the F1F0 ATPase, have not been previously reported to contribute to S. aureus NO· resistance. The most striking finding was that loss of any genes encoding components of the F1F0 ATPase resulted in mutants unable to grow in the presence of NO· or any other condition that inhibits cellular respiration. In addition, these mutants were highly attenuated in murine SSTIs. We show that in S. aureus, the F1F0 ATPase operates in the ATP-hydrolysis mode to extrude protons and contribute to proton-motive force. Loss of efficient proton extrusion in the ΔatpG mutant results in an acidified cytosol. While this acidity is tolerated by respiring cells, enzymes required for fermentation cannot operate efficiently at pH ≤ 7.0 and the ΔatpG mutant cannot thrive. Thus, S. aureus NO· resistance requires a mildly alkaline cytosol, a condition that cannot be achieved without an active F1F0 ATPase enzyme complex.

Regulatory Requirements for Staphylococcus aureus Nitric Oxide Resistance.

UNLABELLED: The ability of Staphylococcus aureus to resist host innate immunity augments the severity and pervasiveness of its pathogenesis. Nitric oxide (NO˙) is an innate immune radical that is critical for the efficient clearance of a wide range of microbial pathogens. Exposure of microbes to NO˙ typically results in growth inhibition and induction of stress regulons. S. aureus, however, induces a metabolic state in response to NO˙ that allows for continued replication and precludes stress regulon induction. The regulatory factors mediating this distinctive response remain largely undefined. Here, we employ a targeted transposon screen and transcriptomics to identify and characterize five regulons essential for NO˙ resistance in S. aureus: three virulence regulons not formerly associated with NO˙ resistance, SarA, CodY, and Rot, as well as two regulons with established roles, Fur and SrrAB. We provide new insights into the contributions of Fur and SrrAB during NO˙ stress and show that the S. aureus ΔsarA mutant, the most sensitive of the newly identified mutants, exhibits metabolic dysfunction and widespread transcriptional dysregulation following NO˙ exposure. Altogether, our results broadly characterize the regulatory requirements for NO˙ resistance in S. aureus and suggest an intriguing overlap between the regulation of NO˙ resistance and virulence in this well-adapted human pathogen. IMPORTANCE: The prolific human pathogen Staphylococcus aureus is uniquely capable of resisting the antimicrobial radical nitric oxide (NO˙), a crucial component of the innate immune response. However, a complete understanding of how S. aureus regulates an effective response to NO˙ is lacking. Here, we implicate three central virulence regulators, SarA, CodY, and Rot, as major players in the S. aureus NO˙ response. Additionally, we elaborate on the contribution of two regulators, SrrAB and Fur, already known to play a crucial role in S. aureus NO˙ resistance. Our study sheds light on a unique facet of S. aureus pathogenicity and demonstrates that the transcriptional response of S. aureus to NO˙ is highly pleiotropic and intrinsically tied to metabolism and virulence regulation.

Commensal microbiota stimulate systemic neutrophil migration through induction of serum amyloid A.

Neutrophils serve critical roles in inflammatory responses to infection and injury, and mechanisms governing their activity represent attractive targets for controlling inflammation. The commensal microbiota is known to regulate the activity of neutrophils and other leucocytes in the intestine, but the systemic impact of the microbiota on neutrophils remains unknown. Here we utilized in vivo imaging in gnotobiotic zebrafish to reveal diverse effects of microbiota colonization on systemic neutrophil development and function. The presence of a microbiota resulted in increased neutrophil number and myeloperoxidase expression, and altered neutrophil localization and migratory behaviours. These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to injury. Genetic analysis identified the microbiota-induced acute phase protein serum amyloid A (Saa) as a host factor mediating microbial stimulation of tissue-specific neutrophil migratory behaviours. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB, and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression. These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signalling axis in mediating neutrophil migratory responses.

A MRSA mystery: how PBP4 and cyclic-di-AMP join forces against ß-lactam antibiotics.

The high-level resistance to next-generation ß-lactams frequently found in Staphylococcus aureus isolates lacking mec, which encodes the transpeptidase PBP2a traditionally associated with methicillin-resistant Staphylococcus aureus (MRSA), has remained incompletely understood for decades. A new study by Lai et al. found that the co-occurrence of mutations in pbp4 and gdpP, which respectively cause increased PBP4-mediated cell wall crosslinking and elevated cyclic-di-AMP levels, produces synergistic ß-lactam resistance rivaling that of PBP2a-producing MRSA (L.-Y. Lai, N. Satishkumar, S. Cardozo, V. Hemmadi, et al., mBio 15:e02889-23. 2024, https://doi.org/10.1128/mbio.02889-23). The combined mutations are sufficient to explain the high-level ß-lactam resistance of some mec-lacking strains, but the mechanism of synergy remains elusive and an avenue for further research. Importantly, the authors establish that co-occurrence of these mutations leads to antibiotic therapy failure in a Caenorhabditis elegans infection model. These results underscore the need to consider this unique and novel ß-lactam resistance mechanism during the clinical diagnosis of MRSA, rather than relying on mec as a diagnostic.

Plant mitochondrial introns as genetic markers - conservation and variation.

Plant genomes are comprised of nuclear, plastid and mitochondrial components characterized by different patterns of inheritance and evolution. Genetic markers from the three genomes provide complementary tools for investigations of inheritance, genetic relationships and phenotypic contributions. Plant mitochondrial genomes are challenging for universal marker development because they are highly variable in terms of size, gene order and intergenic sequences and highly conserved with respect to protein-coding sequences. PCR amplification of introns with primers that anneal to conserved, flanking exons is effective for the development of polymorphic nuclear genome markers. The potential for plant mitochondrial intron polymorphisms to distinguish between congeneric species or intraspecific varieties has not been systematically investigated and is possibly constrained by requirements for intron secondary structure and interactions with co-evolved organelle intron splicing factors. To explore the potential for broadly applicable plant mitochondrial intron markers, PCR primer sets based upon conserved sequences flanking 11 introns common to seven angiosperm species were tested across a range of plant orders. PCR-amplified introns were screened for indel polymorphisms among a group of cross-compatible Citrus species and relatives; two Raphanus sativus mitotypes; representatives of the two Phaseolus vulgaris gene pools; and congeneric pairs of Cynodon, Cenchrus, Solanum, and Vaccinium species. All introns were successfully amplified from each plant entry. Length polymorphisms distinguishable by gel electrophoresis were common among genera but infrequent within genera. Sequencing of three introns amplified from 16 entries identified additional short indel polymorphisms and nucleotide substitutions that separated Citrus, Cynodon, Cenchrus and Vaccinium congeners, but failed to distinguish Solanum congeners or representatives of the Phaseolus vulgaris major gene pools. The ability of primer sets to amplify a wider range of plant species' introns and the presence of intron polymorphisms that distinguish congeners was confirmed by in silico analysis. While mitochondrial intron variation is limited in comparison to nuclear introns, these exon-based primer sets provide robust tools for the amplification of mitochondrial introns across a wide range of plant species wherein useful polymorphisms can be identified.

Design and evaluation of poly-nitrogenous adjuvants capable of potentiating antibiotics in Gram-negative bacteria.

Antibiotic resistance has been a growing public health crisis since the 1980s. Therefore, it is essential not only to continue to develop novel antibiotics but also to develop new methods for overcoming resistance mechanisms in pathogenic bacteria so antibiotics can be reactivated towards these resistant strains. One common cause of antibiotic resistance in Gram-negative bacteria is reduced permeability of the tightly packed, negatively charged lipopolysaccharide outer membrane (OM), which dramatically reduces or even prevents antibiotic accumulation within the cell. Adjuvants that promote passive diffusion through the OM, including phenylalanine-arginine-ß-naphthylamide, tobramycin, and pentamidine, have proven useful in potentiating antibiotics against Gram-negative bacteria. Structural evaluation of these adjuvants, which all include multiple nitrogenous groups, indicates that the entry rules developed for improving antibiotic accumulation in Escherichia coli (EC), could also be used to guide adjuvant development. To this end, a series of structurally simple poly-nitrogenous diphenylsuccinamide compounds have been prepared and evaluated for their ability to potentiate a panel of classic antibiotics in wild-type EC and Pseudomonas aeruginosa (PA). Modest adjuvant activity was observed for all compounds surveyed when co-administered with known antibiotics to inhibit either wild-type EC or PA, and all were able to accumulate in both EC and PA.

Pseudomonas aeruginosa Can Diversify after Host Cell Invasion to Establish Multiple Intracellular Niches.

Within epithelial cells, Pseudomonas aeruginosa depends on its type III secretion system (T3SS) to escape vacuoles and replicate rapidly in the cytosol. Previously, it was assumed that intracellular subpopulations remaining T3SS-negative (and therefore in vacuoles) were destined for degradation in lysosomes, supported by data showing vacuole acidification. Here, we report in both corneal and bronchial human epithelial cells that vacuole-associated bacteria can persist, sometimes in the same cells as cytosolic bacteria. Using a combination of phase-contrast, confocal, and correlative light-electron microscopy (CLEM), we also found they can demonstrate biofilm-associated markers: cdrA and cyclic-di-GMP (c-di-GMP). Vacuolar-associated bacteria, but not their cytosolic counterparts, tolerated the cell-permeable antibiotic ofloxacin. Surprisingly, use of mutants showed that both persistence in vacuoles and ofloxacin tolerance were independent of the biofilm-associated protein CdrA or exopolysaccharides (Psl, Pel, alginate). A T3SS mutant (ΔexsA) unable to escape vacuoles phenocopied vacuole-associated subpopulations in wild-type PAO1-infected cells, with results revealing that epithelial cell death depended upon bacterial viability. Intravital confocal imaging of infected mouse corneas confirmed that P. aeruginosa formed similar intracellular subpopulations within epithelial cells in vivo. Together, these results show that P. aeruginosa differs from other pathogens by diversifying intracellularly into vacuolar and cytosolic subpopulations that both contribute to pathogenesis. Their different gene expression and behavior (e.g., rapid replication versus slow replication/persistence) suggest cooperation favoring both short- and long-term interests and another potential pathway to treatment failure. How this intracellular diversification relates to previously described "acute versus chronic" virulence gene-expression phenotypes of P. aeruginosa remains to be determined. IMPORTANCE Pseudomonas aeruginosa can cause sight- and life-threatening opportunistic infections, and its evolving antibiotic resistance is a growing concern. Most P. aeruginosa strains can invade host cells, presenting a challenge to therapies that do not penetrate host cell membranes. Previously, we showed that the P. aeruginosa type III secretion system (T3SS) plays a pivotal role in survival within epithelial cells, allowing escape from vacuoles, rapid replication in the cytoplasm, and suppression of host cell death. Here, we report the discovery of a novel T3SS-negative subpopulation of intracellular P. aeruginosa within epithelial cells that persist in vacuoles rather than the cytoplasm and that tolerate a cell-permeable antibiotic (ofloxacin) that is able to kill cytosolic bacteria. Classical biofilm-associated markers, although demonstrated by this subpopulation, are not required for vacuolar persistence or antibiotic tolerance. These findings advance our understanding of how P. aeruginosa hijacks host cells, showing that it diversifies into multiple populations with T3SS-negative members enabling persistence while rapid replication is accomplished by more vulnerable T3SS-positive siblings. Intracellular P. aeruginosa persisting and tolerating antibiotics independently of the T3SS or biofilm-associated factors could present additional challenges to development of more effective therapeutics.

Analysis of a COVID-19 Clinical Trial to Emphasize Experimental Design and Quantitative Reasoning in an Introductory Biology Course.

Increasing student exposure to primary literature in early biology coursework can enhance scientific literacy and quantitative reasoning skills. The efficacy of primary literature discussion is heavily impacted by article selection, as student engagement is optimal with material that is topical and has clear relevance to real world issues. During the COVID-19 pandemic, the prevalence of COVID-19-related scientific research in the mainstream media makes it an ideal topic for current discussion in entry-level biology courses. Here, we present an activity developed to facilitate a remote, synchronous discussion of an open access clinical trial publication on the experimental drug remdesivir in the treatment of COVID-19 (Beigel et al., 2020, N Engl J Med https://doi.org/10.1056/nejmoa2007764). The activity, which is amenable to adaptation for other research articles, emphasizes concepts in experimental design, statistical analysis, graphical interpretation, and the structure, content, and organization of typical sections of a primary research article. Importantly, the activity highlights the utility of the classroom response tool Pear Deck, a Google Slides add-on, for creating engaging literature discussions that can be readily adapted to a wide variety of teaching modalities.

Human tear fluid modulates the Pseudomonas aeruginosa transcriptome to alter antibiotic susceptibility.

PURPOSE: Previously, we showed that tear fluid protects corneal epithelial cells against Pseudomonas aeruginosa without suppressing bacterial viability. Here, we studied how tear fluid affects bacterial gene expression. METHODS: RNA-sequencing was used to study the P. aeruginosa transcriptome after tear fluid exposure (5 h, 37 oC). Outcomes were further investigated by biochemical and physiological perturbations to tear fluid and tear-like fluid (TLF) and assessment of bacterial viability following tear/TLF pretreatment and antibiotic exposure. RESULTS: Tear fluid deregulated ~180 P. aeruginosa genes ≥8 fold versus PBS including downregulating lasI, rhlI, qscR (quorum sensing/virulence), oprH, phoP, phoQ (antimicrobial resistance) and arnBCADTEF (polymyxin B resistance). Upregulated genes included algF (biofilm formation) and hemO (iron acquisition). qPCR confirmed tear down-regulation of oprH, phoP and phoQ. Tear fluid pre-treatment increased P. aeruginosa resistance to meropenem ~5-fold (4 µg/ml), but enhanced polymyxin B susceptibility ~180-fold (1 µg/ml), the latter activity reduced by dilution in PBS. Media containing a subset of tear components (TLF) also sensitized bacteria to polymyxin B, but only ~22.5-fold, correlating with TLF/tear fluid Ca2+ and Mg2+ concentrations. Accordingly, phoQ mutants were not sensitized by TLF or tear fluid. Superior activity of tear fluid versus TLF against wild-type P. aeruginosa was heat resistant but proteinase K sensitive. CONCLUSION: P. aeruginosa responds to human tear fluid by upregulating genes associated with bacterial survival and adaptation. Meanwhile, tear fluid down-regulates multiple virulence-associated genes. Tears also utilize divalent cations and heat resistant/proteinase K sensitive component(s) to enhance P. aeruginosa sensitivity to polymyxin B.

Contact lens-related corneal infection: Intrinsic resistance and its compromise.

Contact lenses represent a widely utilized form of vision correction with more than 140 million wearers worldwide. Although generally well-tolerated, contact lenses can cause corneal infection (microbial keratitis), with an approximate annualized incidence ranging from ~2 to ~20 cases per 10,000 wearers, and sometimes resulting in permanent vision loss. Research suggests that the pathogenesis of contact lens-associated microbial keratitis is complex and multifactorial, likely requiring multiple conspiring factors that compromise the intrinsic resistance of a healthy cornea to infection. Here, we outline our perspective of the mechanisms by which contact lens wear sometimes renders the cornea susceptible to infection, focusing primarily on our own research efforts during the past three decades. This has included studies of host factors underlying the constitutive barrier function of the healthy cornea, its response to bacterial challenge when intrinsic resistance is not compromised, pathogen virulence mechanisms, and the effects of contact lens wear that alter the outcome of host-microbe interactions. For almost all of this work, we have utilized the bacterium Pseudomonas aeruginosa because it is the leading cause of lens-related microbial keratitis. While not yet common among corneal isolates, clinical isolates of P. aeruginosa have emerged that are resistant to virtually all currently available antibiotics, leading the United States CDC (Centers for Disease Control) to add P. aeruginosa to its list of most serious threats. Compounding this concern, the development of advanced contact lenses for biosensing and augmented reality, together with the escalating incidence of myopia, could portent an epidemic of vision-threatening corneal infections in the future. Thankfully, technological advances in genomics, proteomics, metabolomics and imaging combined with emerging models of contact lens-associated P. aeruginosa infection hold promise for solving the problem - and possibly life-threatening infections impacting other tissues.

Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells.

Pseudomonas aeruginosa is among bacterial pathogens capable of twitching motility, a form of surface-associated movement dependent on type IV pili (T4P). Previously, we showed that T4P and twitching were required for P. aeruginosa to cause disease in a murine model of corneal infection, to traverse human corneal epithelial multilayers, and to efficiently exit invaded epithelial cells. Here, we used live wide-field fluorescent imaging combined with quantitative image analysis to explore how twitching contributes to epithelial cell egress. Results using time-lapse imaging of cells infected with wild-type PAO1 showed that cytoplasmic bacteria slowly disseminated throughout the cytosol at a median speed of >0.05 µm s-1 while dividing intracellularly. Similar results were obtained with flagellin (fliC) and flagellum assembly (flhA) mutants, thereby excluding swimming, swarming, and sliding as mechanisms. In contrast, pilA mutants (lacking T4P) and pilT mutants (twitching motility defective) appeared stationary and accumulated in expanding aggregates during intracellular division. Transmission electron microscopy confirmed that these mutants were not trapped within membrane-bound cytosolic compartments. For the wild type, dissemination in the cytosol was not prevented by the depolymerization of actin filaments using latrunculin A and/or the disruption of microtubules using nocodazole. Together, these findings illustrate a novel form of intracellular bacterial motility differing from previously described mechanisms in being directly driven by bacterial motility appendages (T4P) and not depending on polymerized host actin or microtubules.IMPORTANCE Host cell invasion can contribute to disease pathogenesis by the opportunistic pathogen Pseudomonas aeruginosa Previously, we showed that the type III secretion system (T3SS) of invasive P. aeruginosa strains modulates cell entry and subsequent escape from vacuolar trafficking to host lysosomes. However, we also showed that mutants lacking either type IV pili (T4P) or T4P-dependent twitching motility (i) were defective in traversing cell multilayers, (ii) caused less pathology in vivo, and (iii) had a reduced capacity to exit invaded cells. Here, we report that after vacuolar escape, intracellular P. aeruginosa can use T4P-dependent twitching motility to disseminate throughout the host cell cytoplasm. We further show that this strategy for intracellular dissemination does not depend on flagellin and resists both host actin and host microtubule disruption. This differs from mechanisms used by previously studied pathogens that utilize either host actin or microtubules for intracellular dissemination independently of microbe motility appendages.

Method for Preparation and Electroporation of S. aureus and S. epidermidis.

For bacterial species that are not known to be naturally competent, such as Staphylococcus aureus and Staphylococcus epidermidis, electroporation is an efficient method for introducing genetic material into the cell. The technique utilizes electrical pulses to transiently permeabilize bacterial cell membranes, which allows for the passage of plasmid DNA across the membranes. Here, we describe methods for preparing electrocompetent S. aureus and S. epidermidis cells and outline a procedure for electroporation of the prepared competent cells.

Expanded Glucose Import Capability Affords Staphylococcus aureus Optimized Glycolytic Flux during Infection.

UNLABELLED: Acquisition of numerous virulence determinants affords Staphylococcus aureus greater pathogenicity than other skin-colonizing staphylococci in humans. Additionally, the metabolic adaptation of S. aureus to nonrespiratory conditions encountered during infection (e.g., hypoxia, nitric oxide, iron chelation) has been implicated as contributing to S. aureus virulence. Specifically, S. aureus has been shown to ferment glycolytic substrates in nonrespiratory environments encountered within the host. Here, we show that S. aureus has acquired unique carbohydrate transporters that facilitate the maximal uptake of host sugars and serve to support nonrespiratory growth in inflamed tissue. The carbohydrate substrates of 11 S. aureus transporters were identified, and at least four of their genes encode S. aureus glucose transporters (glcA, glcB, glcC, and glcU). Moreover, two transporter genes (glcA and glcC) are unique to S. aureus and contribute disproportionately to the nonrespiratory growth of S. aureus on glucose. Targeted inactivation of sugar transporters reduced glucose uptake and attenuated S. aureus in a murine model of skin and soft tissue infections. These data expand the evidence for metabolic adaptation of S. aureus to invasive infection and demonstrate the specific requirement for the fermentation of glucose over all other available carbohydrates. Ultimately, acquisition of foreign genes allows S. aureus to adopt a metabolic strategy resembling that of infiltrating host immune cells: high glycolytic flux coupled to lactate excretion. IMPORTANCE: The bacterial pathogen Staphylococcus aureus causes a wide range of human infections that are costly and difficult to treat. S. aureus differs from closely related commensal staphylococci in its ability to flourish following the invasion of deeper tissue from the skin surface. There, S. aureus primarily uses glucose to grow under respiration-limiting conditions imposed by the immune system. It was previously unclear how S. aureus thrives in this environment when other Staphylococcus species cannot. Our results provide evidence that S. aureus has acquired an expanded repertoire of carbohydrate transporters. In particular, four glucose transporters contribute to efficient S. aureus growth during infection. Thus, S. aureus has evolved to maximize its glucose uptake abilities for enhanced glycolytic flux during tissue invasion. This dependence on glucose acquisition for S. aureus virulence may also explain links between serious infectious complications associated with diabetic patients exhibiting elevated blood glucose levels.