The physiological role of Acinetobacter baumannii DacC is exerted through influencing cell shape, biofilm formation, the fitness of survival and manifesting DD-carboxypeptidase and beta-lactamase dual-enzyme activities.

With the growing threat of drug-resistant Acinetobacter baumannii, there is an urgent need to comprehensively understand the physiology of this nosocomial pathogen. As penicillin-binding proteins are attractive targets for antibacterial therapy, we have tried to explore the physiological roles of two putative DD-carboxypeptidases, viz., DacC and DacD, in A. baumannii. Surprisingly, the deletion of dacC resulted in a reduced growth rate, loss of rod-shaped morphology, reduction in biofilm-forming ability, and enhanced susceptibility towards beta-lactams. In contrast, the deletion of dacD had no such effect. Interestingly, ectopic expression of dacC restored the lost phenotypes. The ∆dacCD mutant showed properties similar to the ∆dacC mutant. Conversely, in vitro enzyme kinetics assessments reveal that DacD is a stronger DD-CPase than DacC. Finally, we conclude that DacC might have DD-CPase and beta-lactamase activities, whereas DacD is a strong DD-CPase.

Molecular Basis of Influence of A501X Mutations in Penicillin-Binding Protein 2 of Neisseria gonorrhoeae Strain 35/02 on Ceftriaxone Resistance.

The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the ß-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the ß3-ß4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.

Activity of aztreonam/avibactam and ceftazidime/avibactam against Enterobacterales with carbapenemase-independent carbapenem resistance.

Enterobacterales with carbapenemase-independent resistance to carbapenems are sometimes selected during therapy and, on rare occasions, cause outbreaks. Most have extended-spectrum or AmpC ß-lactamases, together with changes to permeability or penicillin-binding proteins (PBPs). Newer ß-lactam-ß-lactamase inhibitor combinations may present useful options for infections due to these organisms. Accordingly, Clinical and Laboratory Standards Institute/European Committee on Antimicrobial Susceptibility Testing broth-microdilution was used to measure the minimum inhibitory concentrations (MICs) of ceftazidime/avibactam and aztreonam/avibactam for 51 carbapenemase-negative Enterobacterales with resistance or reduced susceptibility to carbapenems: genomic sequencing of the least-susceptible organisms was also undertaken. MICs of the two avibactam combinations cross-correlated closely, but with fewer MICs (2/51 vs. 10/51) exceeding 8+4 mg/L in the case of ceftazidime/avibactam. Raised MICs for Escherichia coli were associated with PBP3 inserts together with CMY-42 ß-lactamase; correlates among Enterobacter cloacae complex isolates remain elusive, with AmpC and PBP3 sequences found to be species specific. In the case of Klebsiella spp., no MICs exceeding 2 mg/L were seen for either combination. It appears that these avibactam combinations have potential against Enterobacterales with carbapenemase-independent carbapenem resistance or reduced susceptibility, with ceftazidime/avibactam being more reliably active than aztreonam/avibactam.

The peptidoglycan synthase PBP interacts with PLASTID DIVISION2 to promote chloroplast division in Physcomitrium patens.

The peptidoglycan (PG) layer, a core component of the bacterial cell wall, has been retained in the Physcomitrium patens chloroplasts. The PG layer entirely encompasses the P. patens chloroplast, including the division site, but how PG biosynthesis cooperates with the constriction of two envelope membranes at the chloroplast division site remains elusive. Here, focusing on the PG synthase penicillin-binding protein (PBP), we performed cytological and molecular analyses to dissect the mechanism of chloroplast division in P. patens. We showed that PBP, acting in the final step of PG biosynthesis, is likely a chloroplast inner envelope protein that can aggregate at mid-chloroplasts during chloroplast division. Physcomitrium patens had five orthologs of PLASTID DIVISION2 (PDV2), an outer envelope component of the chloroplast division complex. Our data indicated that PpPDV2 proteins interact with PpPBP and are responsible for recruiting PpPBP to the chloroplast division site, in addition to PpDRP5B. Furthermore, we found that PBP deletion and carbenicillin application restrain constriction of the chloroplast division complex, rather than its assembly. This work provides direct molecular evidence for a link between chloroplast division of P. patens and PG biosynthesis and indicates that PG biosynthesis is required for the constriction of the chloroplast division apparatus in P. patens.

Activity of aztreonam/avibactam against metallo-ß-lactamase-producing Enterobacterales from the UK: Impact of penicillin-binding protein-3 inserts and CMY-42 ß-lactamase in Escherichia coli.

Aztreonam/avibactam is being developed on the rationale that aztreonam evades metallo-ß-lactamases (MBLs) whilst avibactam protects aztreonam against co-produced serine ß-lactamases. This study measured the activity of aztreonam/avibactam against MBL-producing Enterobacterales referred to the UK Health Security Agency in 2015, 2017 and 2019. Minimum inhibitory concentrations (MICs) were determined by broth microdilution, and genome sequences were determined with Illumina technology. For Klebsiella and Enterobacter spp. with NDM, IMP or VIM enzymes, the MICs of aztreonam/avibactam were distributed unimodally, with >90% of isolates inhibited at 1+4 mg/L, and all inhibited at 8+4 mg/L. Over 85% of Escherichia coli with NDM carbapenemases were inhibited at 8+4 mg/L, but their MIC distribution was multi-modal with major peaks at 0.12 and 8 mg/L. Forty-eight of 50 NDM E. coli with high aztreonam/avibactam MICs (defined as ≥8 mg/L) had YRIK inserted after amino acid 333 of penicillin-binding protein (PBP)3, or had a YRIN insert plus an acquired AmpC ß-lactamase, commonly CMY-42. Ten of 15 E. coli with moderately raised aztreonam/avibactam MICs (defined as 0.5-4 mg/L) had YRIN inserts without acquired AmpC. Twenty-two of 24 E. coli isolates with normal MICs (defined as 0.03-0.25 mg/L) lacked PBP3 inserts. YRIK inserts were associated with E. coli ST405, and YRIN inserts with ST167; however, many isolates with high or moderately raised MICs were clonally diverse. No substantive MIC distribution shifts occurred across the three survey years; ST405 isolates with YRIK comprised more high-MIC organisms in 2019 compared with earlier years, but the apparent increase lacked significance (P>0.05).

Penicillin-binding protein 1b encoded by mrcB gene mediates the enhancement of biofilm formation by subinhibitory concentrations of cefotaxime in monophasic Salmonella Typhimurium strain SH16SP46.

Development of cefotaxime-resistance and biofilm formation increase the difficulty to prevent and control the infection and contamination of Salmonella, one of the most important foodborne and zoonotic bacterial pathogen. Our previous study observed that 1/8 minimum inhibitory concentration (MIC) of cefotaxime induced the enhancement of biofilm formation and filamentous morphological change by a monophasic Salmonella Typhimurium strain SH16SP46. This study was designed to explore the role of three penicillin-binding proteins (PBPs) in mediating the induction effect of cefotaxime. Three deletion mutants of the genes mrcA, mrcB, and ftsI, encoding the proteins PBP1a, PBP1b, and PBP3, respectively, were constructed using the parental Salmonella strain SH16SP46. Gram staining and scanning electron microscopy showed that these mutants showed normal morphology comparable to the parental strain without cefotaxime treatment. However, under the stress of 1/8 MIC of cefotaxime, the strains WT, ΔmrcA, and ΔftsI, rather than ΔmrcB, exhibited filamentous morphological change. Moreover, cefotaxime treatment significantly enhanced biofilm formation by the strains WT, ΔmrcA, and ΔftsI, but not by the ΔmrcB strain. The complement of mrcB gene in the ΔmrcB strain recovered the enhanced biofilm formation and filamentous morphological change induced by cefotaxime. Our results suggest that PBP1b encoded by mrcB gene may be a binding target of cefotaxime for initiating the effect on Salmonella morphology and biofilm formation. The study will contribute to further understanding of the regulatory mechanism of cefotaxime on Salmonella biofilm formation.

New Insights into Beta-Lactam Resistance of Streptococcus pneumoniae: Serine Protease HtrA Degrades Altered Penicillin-Binding Protein 2x.

Reduced amounts of the essential penicillin-binding protein 2x (PBP2x) were detected in two cefotaxime-resistant Streptococcus pneumoniae laboratory mutants C405 and C606. These mutants contain two or four mutations in the penicillin-binding domain of PBP2x, respectively. The transcription of the pbp2x gene was not affected in both mutants; thus, the reduced PBP2x amounts were likely due to post-transcriptional regulation. The mutants carry a mutation in the histidine protein kinase gene ciaH, resulting in enhanced gene expression mediated by the cognate response regulator CiaR. Deletion of htrA, encoding a serine protease regulated by CiaR, or inactivation of HtrA proteolytic activity showed that HtrA is indeed responsible for PBP2x degradation in both mutants, and that this affects ß-lactam resistance. Depletion of the PBP2xC405 in different genetic backgrounds confirmed that HtrA degrades PBP2xC405. A GFP-PBP2xC405 fusion protein still localized at the septum in the absence of HtrA. The complementation studies in HtrA deletion strains showed that HtrA can be overexpressed in pneumococcal cells to specific levels, depending on the genetic background. Quantitative Western blotting revealed that the PBP2x amount in C405 strain was less than 20% compared to parental strain, suggesting that PBP2x is an abundant protein in S. pneumoniae R6 strain.

PBP Isolation and DD-Carboxypeptidase Assay.

Penicillin-binding proteins (PBPs) share the namesake because of their ability to bind penicillin or any beta-lactam antibiotic. In other words, PBPs are the targets of ß-lactam antibiotics that hold nearly 60% of the global antibiotic market. These enzymes catalyze the final stages of peptidoglycan (PG) biosynthesis by acting as transglycosylases and transpeptidases. PBPs are also involved in PG remodeling by catalyzing DD-carboxypeptidase (DD-CPase) and endopeptidase reactions. Though the cross-linking abilities of PBPs are well known, the process of remodeling is still unclear, thereby drawing attention toward the DD-CPase enzymes. Here, we describe the step-by-step procedures for isolation of the bacterial cell membrane and detection of PBPs in it, followed by the purification of PBPs (DD-CPases) by both ampicillin-affinity and nickel-nitrilotriacetic acid (Ni-NTA) chromatography. The protocols to determine the enzymatic efficiency are also elucidated. The assays are aimed to determine the kinetic parameters for the interaction of the PBP with BOCILLIN, to evaluate its acylation and deacylation rates, and with its peptide substrates, to assess its DD-CPase activity.

On-Chip Isoniazid Exposure of Mycobacterium smegmatis Penicillin-Binding Protein (PBP) Mutant Using Time-Lapse Fluorescent Microscopy.

Antibiotic resistance has been one of the biggest threats to global health. Despite the available prevention and control strategies and efforts in developing new antibiotics, the need remains for effective approaches against antibiotic resistance. Efficient strategies to cope with antimicrobial resistance require a quantitative and deeper understanding of microbial behavior, which can be obtained using different techniques to provide the missing pieces of the current antibiotic-resistance puzzle. Microfluidic-microscopy techniques are among the most promising methods that contribute modernization of traditional assays in microbiology. They provide monitoring and manipulation of cells at micro-scale volumes. Here, we combined population-level, culture-based assays with single-cell resolution, microfluidic-microscopy systems to investigate isoniazid response of Mycobacterium smegmatis penicillin-binding protein (PBP) mutant. This mutant exhibited normal growth in plain medium and sensitivity to stress responses when treated with thermal stress (45 °C), detergent stress (0.1% sodium dodecyl sulfate), acid stress (pH 4.5), and nutrient starvation (1XPBS). The impact of msm0031 transposon insertion on drug-mediated killing was determined for isoniazid (INH, 50 µg/mL), rifampicin (RIF, 200 µg/mL), ethionamide (ETH, 200 µg/mL), and ethambutol (EMB, 5 µg/mL). The PBP mutant demonstrated remarkable isoniazid-killing phenotype in batch culture. Therefore, we hypothesized that single-cell analysis will show increased lysis kinetics and fewer intact cells after drug treatment. However, the single-cell analysis data showed that upon isoniazid exposure, the percentage of the intact PBP mutant cells was 24%, while the percentage of the intact wild-type cells was 4.6%. The PBP mutant cells exhibited decreased cell-lysis profile. Therefore, the traditional culture-based assays were not sufficient to provide insights about the subpopulation of viable but non-culture cells. Consequently, we need more adequate tools to be able to comprehend and fight the antibiotic resistance of bacteria.

Development of an immunoaffinity solid phase microextraction method for the identification of penicillin binding protein 2a.

Methicillin resistant Staphylococcus aureus (MRSA) is a bacterium that is resistant to many antibiotics. Resistance to methicillin is related to production of penicillin binding protein 2a (PBP2a). The currently presented research involves the development of antibody-linked immunoaffinity solid phase microextraction (SPME) sorbents characterized from several aspects that can identify protein PBP2a. The on-sorbent binding constant Kd of monoclonal anti-PBP2a antibody for its antigen protein PBP2a was determined to be 4×10(-10) M. This value was obtained on the basis of the binding curve determined by selective extraction of its antigen PBP2a at different concentrations. The concentration of PBP2a captured by immunoaffinity sorbents was as low as 10ng/mL; lower concentrations could not be tested due to the sensitivity limitation of the LC-MS/MS system available. Surface density was estimated at 59 ng antibody/cm(2). To reduce non-specific binding, especially when the antigen is a protein, bovine serum albumin (BSA) was used to pretreat surfaces. The established immunoaffinity platform technology is expected to provide insights into the development of a practical, specific, sensitive and accurate assay for in vitro and in vivo diagnostics of MRSA.

The differential detection of methicillin-resistant, methicillin-susceptible and borderline oxacillin-resistant Staphylococcus aureus by surface plasmon resonance.

Two hundred fifty Staphylococcus aureus clinical isolates were studied to determine their susceptibilities to ß-lactam antibiotics. Among these isolates, 16 were methicillin-sensitive S. aureus (MSSA), 207 were methicillin-resistant S. aureus (MRSA) and 27 were borderline oxacillin-resistant S. aureus (BORSA). Currently, the reported mechanism of methicillin resistance in S. aureus is the production of a distinctive penicillin binding protein 2a (PBP2a), which exhibits low affinity toward ß-lactams. A surface plasmon resonance biosensor was evaluated for its ability to identify MRSA and to distinguish these strains from MSSA and BORSA, by specifically detecting PBP2a. We found that the system permits label-free, real-time, specific detection of pathogens for concentrations as low as 10 colony forming units/milliliter (CFU/ml), in less than 20 min. This system promises to become a diagnostic tool for bacteria that cause major public concern in clinical settings.