Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine-associated (PASTA) ...kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen
Listeria monocytogenes
, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a β-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of
reoM
restores cell wall stress sensitivities and cytosolic survival defects of a Δ
prkA
mutant to nearly wild-type levels. While a Δ
prkA
mutant is defective for PG synthesis during cell wall stress, a double Δ
reoM
Δ
prkA
mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of
reoM
in a Δ
prkA
background almost fully restored virulence to wild-type levels. However, loss of
reoM
alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant
Staphylococcus aureus
. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA
in vivo
is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence.
ABSTRACT
Invasive Staphylococcus aureus infections are a leading cause of morbidity and mortality in both hospital and community settings, especially with the widespread emergence of virulent and ...multi-drug resistant methicillin-resistant S. aureus strains. There is an urgent and unmet clinical need for non-antibiotic immune-based approaches to treat these infections as the increasing antibiotic resistance is creating a serious threat to public health. However, all vaccination attempts aimed at preventing S. aureus invasive infections have failed in human trials, especially all vaccines aimed at generating high titers of opsonic antibodies against S. aureus surface antigens to facilitate antibody-mediated bacterial clearance. In this review, we summarize the data from humans regarding the immune responses that protect against invasive S. aureus infections as well as host genetic factors and bacterial evasion mechanisms, which are important to consider for the future development of effective and successful vaccines and immunotherapies against invasive S. aureus infections in humans. The evidence presented form the basis for a hypothesis that staphylococcal toxins (including superantigens and pore-forming toxins) are important virulence factors, and targeting the neutralization of these toxins are more likely to provide a therapeutic benefit in contrast to prior vaccine attempts to generate antibodies to facilitate opsonophagocytosis.
This review summarizes the data from humans regarding the immune responses that protect against invasive Staphylococcus aureus infections as well as host genetic factors and bacterial evasion mechanisms, which form the basis for a hypothesis that future vaccines and immune-based therapies that target the neutralization of staphylococcal toxins superantigens and pore-forming toxins are more likely to provide a therapeutic benefit.
Staphylococcus aureus bacteremia continues to be associated with significant morbidity and mortality, despite improvements in diagnostics and management. Persistent infections pose a major challenge ...to clinicians and have been consistently shown to increase the risk of mortality and other infectious complications. S. aureus, while typically not considered an intracellular pathogen, has been proven to utilize an intracellular niche, through several phenotypes including small colony variants, as a means for survival that has been linked to chronic, persistent, and recurrent infections. This intracellular persistence allows for protection from the host immune system and leads to reduced antibiotic efficacy through a variety of mechanisms. These include antimicrobial resistance, tolerance, and/or persistence in S. aureus that contribute to persistent bacteremia. This review will discuss the challenges associated with treating these complicated infections and the various methods that S. aureus uses to persist within the intracellular space.
Vancomycin is commonly prescribed to hospitalized patients. Decades of pharmacokinetic/pharmacodynamic research culminated in recommendations to monitor the ratio of the area under the ...concentration-time curve (AUC) to the minimum inhibitory concentration in order to optimize vancomycin exposure and minimize toxicity in the revised 2020 guidelines. These guideline recommendations are based on limited data without high-quality evidence and limitations in strength. Despite considerable effort placed on vancomycin therapeutic drug monitoring (TDM), clinicians should recognize that the majority of vancomycin use is empiric. Most patients prescribed empiric vancomycin do not require it beyond a few days. For these patients, AUC determinations during the initial days of vancomycin exposure are futile. This added workload may detract from high-level patient care activities. Loading doses likely achieve AUC targets, so AUC monitoring after a loading dose is largely unnecessary for broad application. The excessive vancomycin TDM for decades has been propagated with limitations in evidence, and it should raise caution on contemporary vancomycin TDM recommendations.
Abstract
Background
Patient interleukin (IL)-1β and IL-10 responses early in Staphylococcus aureus bacteremia (SaB) are associated with bacteremia duration and mortality. We hypothesized that these ...responses vary depending on antimicrobial therapy, with particular interest in whether the superiority of β-lactams links to key cytokine pathways.
Methods
Three medical centers included 59 patients with SaB (47 methicillin-resistant S. aureus MRSA, 12 methicillin-sensitive S. aureus MSSA) from 2015–2017. In the first 48 hours, patients were treated with either a β-lactam (n = 24), including oxacillin, cefazolin, or ceftaroline, or a glyco-/lipopeptide (n = 35), that is, vancomycin or daptomycin. Patient sera from days 1, 3, and 7 were assayed for IL-1β and IL-10 by enzyme-linked immunosorbent assay and compared using the Mann-Whitney U test.
Results
On presentation, IL-10 was elevated in mortality (P = .008) and persistent bacteremia (P = .034), while no difference occurred in IL-1β. Regarding treatment groups, IL-1β and IL-10 were similar prior to receiving antibiotic. Patients treated with β-lactam had higher IL-1β on days 3 (median +5.6 pg/mL; P = .007) and 7 (+10.9 pg/mL; P = .016). Ex vivo, addition of the IL-1 receptor antagonist anakinra to whole blood reduced staphylococcal killing, supporting an IL-1β functional significance in SaB clearance. β-lactam–treated patients had sharper declines in IL-10 than vancomycin or daptomycin –treated patients over 7 days.
Conclusions
These data underscore the importance of β-lactams for SaB, including consideration that the adjunctive role of β-lactams for MRSA in select patients helps elicit favorable host cytokine responses.
In this study, we evaluate the host cytokines interleukin (IL)-1β and IL-10 response in the first 7 days of Staphylococcus aureus bacteremia (SaB) treatment. β-lactam therapy resulted in a more favorable host response, underscoring the importance of using β-lactams whenever possible for SaB, including select methicillin-resistant S. aureus patients to help elicit favorable host cytokine response profiles.
Methicillin-resistant Staphylococcus aureus (MRSA) is a serious clinical threat due to innate virulence properties, high infection rates, and the ability to develop resistance to multiple ...antibiotics, including the lipopeptide daptomycin (DAP). The acquisition of DAP resistance (DAP-R) in MRSA has been linked with several characteristic alterations in the cell envelope. Clinical treatment of DAP-R MRSA infections has generally involved DAP-plus-β-lactam combinations, although definable synergy of such combinations varies in a strain-dependent as well as a β-lactam-dependent manner. We investigated distinct β-lactam-induced cell envelope adaptations of nine clinically derived DAP-susceptible (DAP-S)/DAP-R strain pairs following
exposure to a panel of six standard β-lactams (nafcillin, meropenem, cloxacillin, ceftriaxone, cefaclor, or cefoxitin), which differ in their penicillin-binding protein (PBP)-targeting profiles. In general, in both DAP-S and DAP-R strains, exposure to these β-lactams led to (i) a decreased positive surface charge; (ii) decreased cell membrane (CM) fluidity; (iii) increased content and delocalization of anionic phospholipids (i.e., cardiolipin), with delocalization being more pronounced in DAP-R strains; and (iv) increased DAP binding in DAP-S (but not DAP-R) strains. Collectively, these results suggest that β-lactam-induced alterations in at least three major cell envelope phenotypes (surface charge, membrane fluidity, and cardiolipin content) could underlie improved DAP activity, not mediated solely by an increase in DAP binding. (Note that for ease of presentation, we utilize the terminology "DAP-R" instead of "DAP nonsusceptibility.").
The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen,
, is considered ...an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities. Although
essential genes have been identified by transposon sequencing, they have not been prioritized by sensitivity to knockdown or antibiotics. Here, we take a systems biology approach to comprehensively characterize
essential genes using CRISPR interference (CRISPRi). We show that certain essential genes and pathways are acutely sensitive to knockdown, providing a set of vulnerable targets for future therapeutic investigation. Screening our CRISPRi library against last-resort antibiotics uncovered genes and pathways that modulate beta-lactam sensitivity, an unexpected link between NADH dehydrogenase activity and growth inhibition by polymyxins, and anticorrelated phenotypes that may explain synergy between polymyxins and rifamycins. Our study demonstrates the power of systematic genetic approaches to identify vulnerabilities in Gram-negative pathogens and uncovers antibiotic-essential gene interactions that better inform combination therapies.IMPORTANCE
is a hospital-acquired pathogen that is resistant to many common antibiotic treatments. To combat resistant
infections, we need to identify promising therapeutic targets and effective antibiotic combinations. In this study, we comprehensively characterize the genes and pathways that are critical for
viability. We show that genes involved in aerobic metabolism are central to
physiology and may represent appealing drug targets. We also find antibiotic-gene interactions that may impact the efficacy of carbapenems, rifamycins, and polymyxins, providing a new window into how these antibiotics function in mono- and combination therapies. Our studies offer a useful approach for characterizing interactions between drugs and essential genes in pathogens to inform future therapies.
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