Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to next-generation ß-lactams (NGBs) such as methicillin, nafcillin, and oxacillin. Resistance to NGBs, which is alternatively known as broad-spectrum ß-lactam resistance, is classically mediated by PBP2a, a penicillin-binding protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus spp. serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA-deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as methicillin-resistant lacking mec (MRLM), are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs, can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance toward NGBs at levels comparable to those of MRSAs. Our study provides a fresh new perspective about alternative mechanisms of NGB resistance, challenging our current overall understanding of high-level, broad-spectrum ß-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach toward diagnosis and treatment of ß-lactam-resistant S. aureus infections. IMPORTANCE: In Staphylococcus aureus, high-level, broad-spectrum resistance to ß-lactams such as methicillin, also referred to as methicillin resistance, is largely attributed to mecA. This study demonstrates that S. aureus strains that lack mecA but contain mutations that functionally alter PBP4 and GdpP can also mediate high-level, broad-spectrum resistance to ß-lactams. Resistance brought about by the synergistic action of functionally altered PBP4 and GdpP was phenotypically comparable to that displayed by mecA, as seen by increased bacterial survival in the presence of ß-lactams. An analysis of mutations detected in naturally isolated strains of S. aureus revealed that a significant proportion of them had similar pbp4 and GGDEF domain protein containing phosphodiesterase (gdpP) mutations, making this study clinically significant. This study not only identifies important players of non-classical mechanisms of ß-lactam resistance but also indicates reconsideration of current clinical diagnosis and treatment protocols of S. aureus infections.

Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by Methicillin-Resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to Next Generation ß-lactams (NGB) such as Methicillin, Nafcillin, Oxacillin etc. Resistance to NGBs, which is alternatively known as broad-spectrum ß-lactam resistance is classically mediated by PBP2a, a Penicillin-Binding Protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as Methicillin-Resistant Lacking mec (MRLM) are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance towards NGBs at levels comparable to that of MRSAs. Our study, provides a fresh new perspective about alternative mechanisms of NGBs resistance, challenging our current overall understanding of high-level, broad-spectrum ß-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach towards diagnosis and treatment of ß-lactam resistant S. aureus infections.

Potential Use of Chat GPT in Global Warming.

Climate change is a major global challenge that requires the integration of many different scientific disciplines, including atmospheric science, oceanography, and ecology. The complexity and scale of the problem require sophisticated tools and techniques to understand, model, and project future climate conditions. Artificial intelligence and natural language processing technologies, such as ChatGPT, have the potential to play a critical role in advancing our understanding of climate change and improving the accuracy of climate projections. ChatGPT can be used in a variety of ways to aid climate research, including in model parameterization, data analysis and interpretation, scenario generation, and model evaluation. This technology provides researchers and policy-makers with a powerful tool for generating and analyzing different climate scenarios based on a wide range of data inputs, and for improving the accuracy of climate projections. The author acknowledges asking chatGPT questions regarding its uses for Climate Change Research. Some of the uses that it states are possible now and some are potentials for the future. The author has analyzed and edited the replies of chat GPT.

Role of Chat GPT in Public Health.

ChatGPT, a language model developed by OpenAI, has the potential to play a role in public health. With its ability to generate human-like text based on large amounts of data, ChatGPT has the potential to support individuals and communities in making informed decisions about their health (Panch et al. Lancet Digit Health 1:e13-e14, 2019; Baclic et al. Canada Commun Dis Rep 46.6:161, 2020). However, as with any technology, there are limitations and challenges to consider when using ChatGPT in public health. In this overview, we will examine the potential uses of ChatGPT in public health, as well as the advantages and disadvantages of its use.

Structural basis of broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Broad-spectrum ß-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden1,2. In clinical strains, resistance is largely controlled by BlaR13, a receptor that senses ß-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain4. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (ß-lactamase PC1) and mecA (ß-lactam-resistant cell-wall transpeptidase PBP2a) expression3-7. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously8. Cryo-electron microscopy structures of BlaR1-the wild type and an autocleavage-deficient F284A mutant, with or without ß-lactam-reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from ß-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action-in this case, antibiotic resistance-through the direct cleavage of a repressor.

A Nonclassical Mechanism of ß-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus and Its Effect on Virulence.

Methicillin-resistant Staphylococcus aureus (MRSA) is a group of pathogenic bacteria that are infamously resistant to ß-lactam antibiotics, a property attributed to the mecA gene. Recent studies have reported that mutations associated with the promoter region of pbp4 demonstrated high levels of ß-lactam resistance, suggesting the role of PBP4 as an important non-mecA mediator of ß-lactam resistance. The pbp4-promoter-associated mutations have been detected in strains with or without mecA. Our previous studies that were carried out in strains devoid of mecA described that pbp4-promoter-associated mutations lead to PBP4 overexpression and ß-lactam resistance. In this study, by introducing various pbp4-promoter-associated mutations in the genome of a MRSA strain, we demonstrate that PBP4 overexpression can supplement mecA-associated resistance in S. aureus and can lead to increased ß-lactam resistance. The promoter and regulatory region of pbp4 is shared with a divergently transcribed gene, abcA, which encodes a multidrug exporter. We demonstrate that the promoter mutations caused an upregulation of pbp4 and downregulation of abcA, confirming that the resistant phenotype is associated with PBP4 overexpression. PBP4 has also been associated with staphylococcal pathogenesis, however, its exact role remains unclear. Using a Caenorhabditis elegans model, we demonstrate that strains having increased PBP4 expression are less virulent than wild-type strains, suggesting that ß-lactam resistance mediated via PBP4 likely comes at the cost of virulence. IMPORTANCE Our study demonstrates the ability of PBP4 to be an important mediator of ß-lactam resistance in not only methicillin-susceptible Staphylococcus aureus (MSSA) background strains as previously demonstrated but also in MRSA strains. When present together, PBP2a and PBP4 overexpression can produce increased levels of ß-lactam resistance, causing complications in treatment. Thus, this study suggests the importance of monitoring PBP4-associated resistance in clinical settings, as well as understanding the mechanistic basis of associated resistance, so that treatments targeting PBP4 may be developed. This study also demonstrates that S. aureus strains with increased PBP4 expression are less pathogenic, providing important hints about the role of PBP4 in S. aureus resistance and pathogenesis.

In Vivo Detection of Cyclic-di-AMP in Staphylococcus aureus.

Cyclic-di-AMP (CDA) is a signaling molecule that controls various cellular functions including antibiotic tolerance and osmoregulation in Staphylococcus aureus (S. aureus). In this study, we developed a novel biosensor (bsuO P6-4) for in vivo detection of CDA in S. aureus. The fluorescent biosensor is based on a natural CDA riboswitch from Bacillus subtilis connected at its P6 stem to the dye-binding aptamer Spinach. Our study showed that bsuO P6-4 could detect a wide concentration range of CDA in both laboratory and clinical strains, making it suitable for use in both basic and clinical research applications.

Loss of GdpP Function in Staphylococcus aureus Leads to ß-Lactam Tolerance and Enhanced Evolution of ß-Lactam Resistance.

Infections caused by Staphylococcus aureus are a leading cause of mortality. Treating infections caused by S. aureus is difficult due to resistance against most traditional antibiotics, including ß-lactams. We previously reported the presence of mutations in gdpP among S. aureus strains that were obtained by serial passaging in ß-lactam drugs. Similar mutations have recently been reported in natural S. aureus isolates that are either nonsusceptible or resistant to ß-lactam antibiotics. gdpP codes for a phosphodiesterase that cleaves cyclic-di-AMP (CDA), a newly discovered second messenger. In this study, we sought to identify the role of gdpP in ß-lactam resistance in S. aureus. Our results showed that gdpP-associated mutations caused loss of phosphodiesterase function, leading to increased CDA accumulation in the bacterial cytosol. Deletion of gdpP led to an enhanced ability of the bacteria to withstand a ß-lactam challenge (2 to 3 log increase in bacterial CFU) by promoting tolerance without enhancing MICs of ß-lactam antibiotics. Our results demonstrated that increased drug tolerance due to loss of GdpP function can provide a selective advantage in acquisition of high-level ß-lactam resistance. Loss of GdpP function thus increases tolerance to ß-lactams that can lead to its therapy failure and can permit ß-lactam resistance to occur more readily.

PBP4-mediated ß-lactam resistance among clinical strains of Staphylococcus aureus.

BACKGROUND: PBP4, a low-molecular-weight PBP in Staphylococcus aureus, is not considered to be a classical mediator of ß-lactam resistance. Previous studies carried out by our group with laboratory strains of S. aureus demonstrated the ability of PBP4 to produce ß-lactam resistance through mutations associated with the pbp4 promoter and/or gene. Recent studies of ß-lactam-resistant clinical isolates of S. aureus have reported similar mutations associated with pbp4. OBJECTIVES: To determine if pbp4-associated mutations reported among clinical strains of S. aureus mediate ß-lactam resistance. METHODS: The pbp4 promoters and genes bearing mutations from clinical isolates were cloned into a heterologous host. Reporter, growth and Bocillin assays were performed to assess their role in ß-lactam resistance. X-ray crystallography was used to obtain acyl-enzyme intermediate structures of the WT and mutant PBP4 with nafcillin and cefoxitin. RESULTS: Of the five strains that contained pbp4 promoter mutations, three strains exhibited enhanced expression of PBP4. The R200L mutation in pbp4 resulted in increased survival in the presence of the ß-lactams nafcillin and cefoxitin. Further, introduction of either a promoter or a gene mutation into the genome of a WT host increased the ability of the strains to resist the action of ß-lactams. The four high-resolution X-ray structures presented demonstrate the binding pose of the ß-lactams tested and provide hints for further drug development. CONCLUSIONS: Mutations associated with the pbp4 promoter and pbp4 gene altered protein activity and mediated ß-lactam resistance among the clinically isolated strains that were studied.

Structural analysis of avibactam-mediated activation of the bla and mec divergons in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections cause significant mortality and morbidity globally. MRSA resistance to ß-lactam antibiotics is mediated by two divergons that control levels of a ß-lactamase, PC1, and a penicillin-binding protein poorly acylated by ß-lactam antibiotics, PBP2a. Expression of genes encoding these proteins is controlled by two integral membrane proteins, BlaR1 and MecR1, which both have an extracellular ß-lactam-binding sensor domain. Here, we solved the X-ray crystallographic structures of the BlaR1 and MecR1 sensor domains in complex with avibactam, a diazabicyclooctane ß-lactamase inhibitor at 1.6-2.0 Å resolution. Additionally, we show that S. aureus SF8300, a clinically relevant strain from the USA300 clone of MRSA, responds to avibactam by up-regulating the expression of the blaZ and pbp2a antibiotic-resistance genes, encoding PC1 and PBP2a, respectively. The BlaR1-avibactam structure of the carbamoyl-enzyme intermediate revealed that avibactam is bound to the active-site serine in two orientations ∼180° to each other. Although a physiological role of the observed alternative pose remains to be validated, our structural results hint at the presence of a secondary sulfate-binding pocket that could be exploited in the design of future inhibitors of BlaR1/MecR1 sensor domains or the structurally similar class D ß-lactamases. The MecR1-avibactam structure adopted a singular avibactam orientation similar to one of the two states observed in the BlaR1-avibactam structure. Given avibactam up-regulates expression of blaZ and pbp2a antibiotic resistance genes, we suggest further consideration and research is needed to explore what effects administering ß-lactam-avibactam combinations have on treating MRSA infections.

Stp1 Loss of Function Promotes ß-Lactam Resistance in Staphylococcus aureus That Is Independent of Classical Genes.

ß-Lactam resistance in Staphylococcus aureus limits treatment options. Stp1 and Stk1, a serine-threonine phosphatase and kinase, respectively, mediate serine-threonine kinase (STK) signaling. Loss-of-function point mutations in stp1 were detected among laboratory-passaged ß-lactam-resistant S. aureus strains lacking mecA and blaZ, the major determinants of ß-lactam resistance in the bacteria. Loss of Stp1 function facilitates ß-lactam resistance of the bacteria.

Structural and kinetic analyses of penicillin-binding protein 4 (PBP4)-mediated antibiotic resistance in Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) causes serious community-acquired and nosocomial infections worldwide. MRSA strains are resistant to a variety of antibiotics, including the classic penicillin and cephalosporin classes of ß-lactams, making them intractable to treatment. Although ß-lactam resistance in MRSA has been ascribed to the acquisition and activity of penicillin-binding protein 2a (PBP2a, encoded by mecA), it has recently been observed that resistance can also be mediated by penicillin-binding protein 4 (PBP4). Previously, we have shown that broad-spectrum ß-lactam resistance can arise following serial passaging of a mecA-negative COL strain of S. aureus, creating the CRB strain. This strain has two missense mutations in pbp4 and a mutation in the pbp4 promoter, both of which play an instrumental role in ß-lactam resistance. To better understand PBP4's role in resistance, here we have characterized its kinetics and structure with clinically relevant ß-lactam antibiotics. We present the first crystallographic PBP4 structures of apo and acyl-enzyme intermediate forms complexed with three late-generation ß-lactam antibiotics: ceftobiprole, ceftaroline, and nafcillin. In parallel, we characterized the structural and kinetic effects of the PBP4 mutations present in the CRB strain. Localized within the transpeptidase active-site cleft, the two substitutions appear to have different effects depending on the drug. With ceftobiprole, the missense mutations impaired the Km value 150-fold, decreasing the proportion of inhibited PBP4. However, ceftaroline resistance appeared to be mediated by other factors, possibly including mutation of the pbp4 promoter. Our findings provide evidence that S. aureus CRB has at least two PBP4-mediated resistance mechanisms.

PBP4: A New Perspective on Staphylococcus aureus ß-Lactam Resistance.

ß-lactam antibiotics are excellent drugs for treatment of staphylococcal infections, due to their superior efficacy and safety compared to other drugs. Effectiveness of ß-lactams is severely compromised due to resistance, which is widespread among clinical strains of Staphylococcus aureus. ß-lactams inhibit bacterial cells by binding to penicillin binding proteins (PBPs), which perform the penultimate steps of bacterial cell wall synthesis. Among PBPs of S. aureus, PBP2a has received the most attention for the past several decades due to its preeminent role in conferring both high-level and broad-spectrum resistance to the entire class of ß-lactam drugs. Studies on PBP2a have thus unraveled incredible details of its mechanism of action. We have recently identified that an uncanonical, low molecular weight PBP of S. aureus, PBP4, can also provide high-level and broad-spectrum resistance to the entire class of ß-lactam drugs at a level similar to that of PBP2a. The role of PBP4 has typically been considered not so important for ß-lactam resistance of S. aureus, and as a result its mode of action remains largely unknown. In this article, we review our current knowledge of PBP4 mediating ß-lactam resistance in S. aureus.

PBP4 activity and its overexpression are necessary for PBP4-mediated high-level ß-lactam resistance.

Background: PBP4 is typically considered unimportant for conferring high-level ß-lactam resistance in Staphylococcus aureus. Mutations in PBP4 have been associated with ß-lactam non-susceptibility among natural strains of S. aureus. We have previously shown that PBP4 can mediate high-level ß-lactam resistance in laboratory-generated strains passaged in ß-lactam antibiotics. Mutations in the pbp4 promoter that up-regulate its expression and missense mutations that surround PBP4's active site were detected in high frequencies among passaged strains, suggesting PBP4 plays a key role in resistance. How these mutations participate in PBP4's ability to provide high-level ß-lactam resistance is unknown. Objectives: To determine whether enzymatic activity of PBP4 is required for high-level ß-lactam resistance and to investigate how the pbp4-associated mutations provide ß-lactam resistance. Methods: The catalytic activity of PBP4 was disabled through introduction of a serine to alanine point mutation in its active site (Ser-75→Ala) in a representative and well-studied passaged strain, CRB. pbp4 promoter and missense mutations detected in CRB were reconstituted in a WT strain individually and in combination. ß-Lactam resistance of the resultant strains was evaluated by population analysis. Bacterial peptidoglycan composition of the pbp4 mutants was evaluated with and without antibiotic treatment using LC. Results: PBP4 inactivation imparted complete ß-lactam susceptibility of CRB. Reconstitution of PBP4 missense mutations alone did not impart ß-lactam resistance, but did so in synergism with pbp4 promoter mutation. A similar synergistic interaction of pbp4 mutations was observed in enhanced peptidoglycan cross-linking upon antibiotic treatment. Conclusions: PBP4's activity and overexpression both contribute to high-level ß-lactam resistance.

PBP4 Mediates ß-Lactam Resistance by Altered Function.

Penicillin binding protein 4 (PBP4) can provide high-level ß-lactam resistance in Staphylococcus aureus A series of missense and promoter mutations associated with pbp4 were detected in strains that displayed high-level resistance. We show here that the missense mutations facilitate the ß-lactam resistance mediated by PBP4 and the promoter mutations lead to overexpression of pbp4 Our results also suggest a cooperative interplay among PBPs for ß-lactam resistance.

High-Level Resistance of Staphylococcus aureus to ß-Lactam Antibiotics Mediated by Penicillin-Binding Protein 4 (PBP4).

Penicillin-binding protein 4 (PBP4), a nonessential, low-molecular-weight penicillin-binding protein of Staphylococcus aureus, has been implicated in low-level resistance to ß-lactam antibiotics, although the mechanism is unknown. Mutations in PBP4 and its promoter were identified in a laboratory-generated mutant strain, CRB, which expresses high-level resistance to ß-lactams, including resistance to the new-generation cephalosporins active against methicillin-resistant strains of S. aureus These mutations did not appreciably alter the ß-lactam antibiotic binding affinity of purified recombinant mutant PBP4 compared to that of wild-type PBP4. Compared to the susceptible parent strain, COLnex, the CRB strain produces a highly cross-linked cell wall peptidoglycan, indicative of increased transpeptidase activity. The pbp4 promoter mutation of CRB was associated with greatly increased amounts of PBP4 in membranes compared to those in the COLnex parent. Replacement of the native promoter of COLnex with the mutant promoter of CRB resulted in increased amounts of PBP4 in membranes and a highly cross-linked cell wall. PBP4 can be repurposed to provide essential transpeptidase activity in vivo and confer high-level resistance to ß-lactam antibiotics, such as ceftobiprole and ceftaroline.