Tracing the origin of NDM-1-producing and extensively drug-resistant Pseudomonas aeruginosa ST357 in the Netherlands.

BACKGROUND: In the hospital environment, carbapenemase-producing Pseudomonas aeruginosa (CPPA) may lead to fatal patient infections. However, the transmission routes of CPPA often remain unknown. Therefore, this case study aimed to trace the origin of CPPA ST357, which caused a hospital-acquired pneumonia in a repatriated critically ill patient suffering from Guillain-Barré Syndrome in 2023. METHODS: Antimicrobial susceptibility of the CPPA isolate for 30 single and combination therapies was determined by disk-diffusion, Etest or broth microdilution. Whole-genome sequencing was performed for three case CPPA isolates (one patient and two sinks) and four distinct CPPA ST357 patient isolates received in the Dutch CPPA surveillance program. Furthermore, 193 international P. aeruginosa ST357 assemblies were collected via three genome repositories and analyzed using whole-genome multi-locus sequence typing in combination with antimicrobial resistance gene (ARG) characterization. RESULTS: A Dutch patient who carried NDM-1-producing CPPA was transferred from Kenya to the Netherlands, with subsequent dissemination of CPPA isolates to the local sinks within a month after admission. The CPPA case isolates presented an extensively drug-resistant phenotype, with susceptibility only for colistin and cefiderocol-fosfomycin. Phylogenetic analysis showed considerable variation in allelic distances (mean = 150, max = 527 alleles) among the ST357 isolates from Asia (n = 92), Europe (n = 58), Africa (n = 21), America (n = 16), Oceania (n = 2) and unregistered regions (n = 4). However, the case isolates (n = 3) and additional Dutch patient surveillance program isolates (n = 2) were located in a sub-clade of isolates from Kenya (n = 17; varying 15-49 alleles), the United States (n = 7; 21-115 alleles) and other countries (n = 6; 14-121 alleles). This was consistent with previous hospitalization in Kenya of 2/3 Dutch patients. Additionally, over half of the isolates (20/35) in this sub-clade presented an identical resistome with 9/17 Kenyan, 5/5 Dutch, 4/7 United States and 2/6 other countries, which were characterized by the blaNDM-1, aph(3')-VI, ARR-3 and cmlA1 ARGs. CONCLUSION: This study presents an extensively-drug resistant subclone of NDM-producing P. aeruginosa ST357 with a unique resistome which was introduced to the Netherlands via repatriation of critically ill patients from Kenya. Therefore, the monitoring of repatriated patients for CPPA in conjunction with vigilance for the risk of environmental contamination is advisable to detect and prevent further dissemination.

The Secreted Aminopeptidase of Pseudomonas aeruginosa (PaAP).

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in compromised hosts. P. aeruginosa infections are difficult to treat because of the inherent ability of the bacteria to develop antibiotic resistance, secrete a variety of virulence factors, and form biofilms. The secreted aminopeptidase (PaAP) is an emerging virulence factor, key in providing essential low molecular weight nutrients and a cardinal modulator of biofilm development. PaAP is therefore a new potential target for therapy of P. aeruginosa infections. The present review summarizes the current knowledge of PaAP, with special emphasis on its biochemical and enzymatic properties, activation mechanism, biological roles, regulation, and structure. Recently developed specific inhibitors and their potential as adjuncts in the treatment of P. aeruginosa infections are also described.

Molecular characteristics and evaluation of the phenotypic detection of carbapenemases among Enterobacterales and Pseudomonas via whole genome sequencing.

Background/purposes: The continuously increasing carbapenem resistance within Enterobacterales and Pseudomonas poses a threat to public health, nevertheless, the molecular characteristics of which in southern China still remain limited. And carbapenemase identification is a key factor in effective early therapy of carbapenem-resistant bacteria infections. We aimed to determine the molecular characteristics of these pathogens and compare commercial combined disc tests (CDTs) with the modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) in detecting and distinguishing carbapenemases using whole genome sequencing (WGS). Methods: A total of 78 Enterobacterales, 30 Pseudomonas were obtained from two tertiary hospitals in southern China. Susceptibility tests were conducted using an automated VITEK2 compact system with confirmation via the Kirby-Bauer method. The WGS was conducted on all clinical isolates and the molecular characteristics were analyzed by screening the whole genome sequences. CDTs with or without cloxacillin, mCIM, and eCIM, were performed and compared by taking WGS results as the benchmark. Results: A total of 103 carbapenem non-susceptible and 5 carbapenem susceptible bacteria were determined, with Klebsiella pneumoniae (42.7%), Pseudomonas aeruginosa (23.3%) and Escherichia coli (18.4%) being most prevalent. Carbapenemase genes were detected in 58 (56.3%) of the 103 carbapenem-non-susceptible clinical isolates, including 46 NDM, 6 KPC, 3 IMP, 1 IPM+VIM,1NDM+KPC, and 1 OXA-181. Carbapenemase-producing isolates were detected more frequently in Enterobacterales (76.3%). Among K. pneumoniae, the major sequence types were st307 and st11, while among E. coli and P. aeruginosa, the most prevalent ones were st410 and st242 respectively. For carbapenemase detection in Enterobacterales, the mCIM method achieved 100.00% (95% CI, 92.13-100.00%) sensitivity and 94.44% (70.63-99.71%) specificity (kappa, 0.96); for Pseudomonas, detection sensitivity was 100% (5.46-100.00%), and 100% (84.50-100.00%) specificity (kappa, 0.65). Commercial CDT carbapenemase detection sensitivity for Enterobacterales was 96.49% (86.84-99.39%), and 95.24% (74.13-99.75%) specificity (kappa, 0.90); for Pseudomonas, carbapenemase detection sensitivity was 100.00% (5.46-100.00%) and 37.93% (21.30-57.64%) specificity (kappa, 0.04). When cloxacillin testing was added, CDT specificity reached 84.61% (64.27-94.95%). Conclusion: The molecular epidemiology of carbapenem-non-susceptible isolates from pediatric patients in Southern China exhibited distinctive characteristics. Both the mCIM-eCIM combination and CDT methods effectively detected and differentiated carbapenemases among Enterobacterales isolates, and the former performed better than CDT among Pseudomonas.

Implication of Molecular Constraints Facilitating the Functional Evolution of Pseudomonas aeruginosa KPR2 into a Versatile α-Keto-Acid Reductase.

Theoretical concepts linking the structure, function, and evolution of a protein, while often intuitive, necessitate validation through investigations in real-world systems. Our study empirically explores the evolutionary implications of multiple gene copies in an organism by shedding light on the structure-function modulations observed in Pseudomonas aeruginosa's second copy of ketopantoate reductase (PaKPR2). We demonstrated with two apo structures that the typical active site cleft of the protein transforms into a two-sided pocket where a molecular gate made up of two residues controls the substrate entry site, resulting in its inactivity toward the natural substrate ketopantoate. Strikingly, this structural modification made the protein active against several important α-keto-acid substrates with varied efficiency. Structural constraints at the binding site for this altered functional trait were analyzed with two binary complexes that show the conserved residue microenvironment faces restricted movements due to domain closure. Finally, its mechanistic highlights gathered from a ternary complex structure help in delineating the molecular perspectives behind its kinetic cooperativity toward these broad range of substrates. Detailed structural characteristics of the protein presented here also identified four key amino acid residues responsible for its versatile α-keto-acid reductase activity, which can be further modified to improve its functional properties through protein engineering.

Purification of a chemotherapeutic agent, L-Arginase, from Pseudomonas aeruginosa isolated from soil.

INTRODUCTION: L. arginase refers to the enzyme arginase found in the genus Lactobacillus, it plays a crucial role in the urea cycle, and has implications in various biological applications. This study aimed to purify arginase from Pseudomonas aeruginosa, isolated from soil, and apply it as an anticancer. METHODOLOGY: 28 soil samples of P. aeruginosa were collected from different places of Baghdad, and rice lands in Najaf and Diwaniyah governorates. Different standard laboratory and biochemical assays, and Vitik system were used in diagnosis and growth of arginase enzyme under certain pH, temperature, incubation period. RESULTS: The purified enzyme was precipitated by ammonium sulfite (60-80%), dialyses bag 8000-1000KD, ion exchange by DEAE cellulose and sephadex G100 in gel filtration. Cytotoxicity of arginase against breast t cancer AJM-13 and rat embryo fibroblast REF normal cell line was evaluated for (48 and 72 hours). The inhibition rate increased in the low concentration of abnormal cell (AMJ-13) while decreased in the normal cell (REF), this study takes different concentration (0.392-12.5mg/mL), and low concentration (1562-0.048 mg/mL), the result in high concentration was IR 54.7% during 72 hours for AJM-13 and 14.3% for REF in the same time, while the low concentration was IR 91% in the 1562 mg/mL in the AMJ-13, and 51% in ERF, LD50 of arginase enzyme was 0.781 mg/mL that 41% during 72 hours for ERF, its save to normal cells. CONCLUSIONS: Arginase enzyme, at low concentrations, may have an inhibitory effect on cancer cells, and simultaneously, protect normal cell lines.

Substrate Characterization for Hydrolysis of Multiple Types of Aromatic Esters by Promiscuous Aminopeptidases.

Synthetic aromatic esters, widely employed in agriculture, food, and chemical industries, have become emerging environmental pollutants due to their strong hydrophobicity and poor bioavailability. This study attempted to address this issue by extracellularly expressing the promiscuous aminopeptidase (Aps) from Pseudomonas aeruginosa GF31 in B. subtilis, achieving an impressive enzyme activity of 13.7 U/mg. Notably, we have demonstrated, for the first time, the Aps-mediated degradation of diverse aromatic esters, including but not limited to pyrethroids, phthalates, and parabens. A biochemical characterization of Aps reveals its esterase properties and a broader spectrum of substrate profiles. The degradation rates of p-nitrobenzene esters (p-NB) with different side chain structures vary under the action of Aps, showing a preference for substrates with relatively longer alkyl side chains. The structure-dependent degradability aligns well with the binding energies between Aps and p-NB. Molecular docking and enzyme-substrate interaction elucidate that hydrogen bonding, hydrophobic interactions, and π-π stacking collectively stabilize the enzyme-substrate conformation, promoting substrate hydrolysis. These findings provide new insights into the enzymatic degradation of aromatic ester pollutants, laying a foundation for the further development and modification of promiscuous enzymes.

Biosynthesis of a clickable pyoverdine via in vivo enzyme engineering of an adenylation domain.

The engineering of non ribosomal peptide synthetases (NRPS) for new substrate specificity is a potent strategy to incorporate non-canonical amino acids into peptide sequences, thereby creating peptide diversity and broadening applications. The non-ribosomal peptide pyoverdine is the primary siderophore produced by Pseudomonas aeruginosa and holds biomedical promise in diagnosis, bio-imaging and antibiotic vectorization. We engineered the adenylation domain of PvdD, the terminal NRPS in pyoverdine biosynthesis, to accept a functionalized amino acid. Guided by molecular modeling, we rationally designed mutants of P. aeruginosa with mutations at two positions in the active site. A single amino acid change results in the successful incorporation of an azido-L-homoalanine leading to the synthesis of a new pyoverdine analog, functionalized with an azide function. We further demonstrated that copper free click chemistry is efficient on the functionalized pyoverdine and that the conjugated siderophore retains the iron chelation properties and its capacity to be recognized and transported by P. aeruginosa. The production of clickable pyoverdine holds substantial biotechnological significance, paving the way for numerous downstream applications.

The Human Paraoxonase 2: An Optimized Procedure for Refolding and Stabilization Facilitates Enzyme Analyses and a Proteomics Approach.

The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to pon2 polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of Pseudomonas aeruginosa (PAO1) were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.

Ser/Thr protein kinase Stk1 phosphorylates the key transcriptional regulator AlgR to modulate virulence and resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections worldwide and has emerged as a serious public health threat, due in large part to its multiple virulence factors and remarkable resistance capabilities. Stk1, a eukaryotic-type Ser/Thr protein kinase, has been shown in our previous work to be involved in the regulation of several signalling pathways and biological processes. Here, we demonstrate that deletion of stk1 leads to alterations in several virulence- and resistance-related physiological functions, including reduced pyocyanin and pyoverdine production, attenuated twitching motility, and enhanced biofilm production, extracellular polysaccharide secretion, and antibiotic resistance. Moreover, we identified AlgR, an important transcriptional regulator, as a substrate for Stk1, with its phosphorylation at the Ser143 site catalysed by Stk1. Intriguingly, both the deletion of stk1 and the mutation of Ser143 of AlgR to Ala result in similar changes in the above-mentioned physiological functions. Furthermore, assays of algR expression in these strains suggest that changes in the phosphorylation state of AlgR, rather than its expression level, underlie changes in these physiological functions. These findings uncover Stk1-mediated phosphorylation of AlgR as an important mechanism for regulating virulence and resistance in P. aeruginosa.

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 µM) include PBPs (PBP1a, KD = 0.07 µM; PBP5 = 0.4 µM); other lytic transglycosylases (SltB2, KD = 0.09 µM; RlpA, KD = 0.4 µM); a type VI secretion system effector (Tse5, KD = 0.3 µM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 µM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.

Genomic insights into NDM-1-producing Pseudomonas aeruginosa: Current status in a Bulgarian tertiary hospital and on the Balkans.

The present study aimed to explore the genomic characteristics of eight New Delhi metallo-ß-lactamase-1 (NDM-1)-producing carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from a Bulgarian tertiary hospital (2021-2023) in comparison to blaNDM-1-positive strains originating from the Balkans. Antimicrobial susceptibility testing, phenotypic assays for carbapenemase activity, PCR screening, whole-genome sequencing (WGS), and phylogenomic analysis were performed. Seven of the CRPA isolates investigated (Minimum inhibitory concentration values of imipenem and meropenem >32 mg L-1) were also resistant to piperacillin-tazobactam, ceftazidime, ceftazidime-avibactam, cefepime, ceftolozane-tazobactam, amikacin, tobramycin, ciprofloxacin, and levofloxacin, but were susceptible to colistin (0.5-2 mg L-1) and cefiderocol (0.25-1 mg L-1). The P. aeruginosa Pae57 isolate (designated Pae57) remained susceptible to aminoglycosides as well. WGS uncovered the co-existence of blaNDM-1 and blaGES-1. The isolates belonged to the ST654 high-risk clone, except for Pae57 (ST611). Alignment against reference sequences revealed the presence of a Tn21 transposon harboring bleMBL-blaNDM-1-ISAba125. It was similar to that found in the P. aeruginosa ST654 NDM1_1 strain (GCA_020404785.1) from Serbia. Phylogenomic analysis of our isolates indicated that seven of them (ST654) differed from each other in no more than 44 single-nucleotide polymorphisms (SNPs). Pae57 (ST611) was strikingly different (>21,700 SNPs) compared to all Balkan strains. In conclusion, to our knowledge this is the first report of blaNDM-1-positive P. aeruginosa ST611 isolation, which indicates the transmission dynamics of this determinant between high-risk and potentially high-risk P. aeruginosa clones. Obtained results unveil the dissemination of clonally related NDM-1-producing P. aeruginosa strains in the monitored hospital for approximately a 2-year period.

Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria.

RNA helicases-central enzymes in RNA metabolism-often feature intrinsically disordered regions (IDRs) that enable phase separation and complex molecular interactions. In the bacterial pathogen Pseudomonas aeruginosa, the non-redundant RhlE1 and RhlE2 RNA helicases share a conserved REC catalytic core but differ in C-terminal IDRs. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both IDRs facilitate RNA binding and phase separation, localizing proteins in cytoplasmic clusters. However, RhlE2 IDR is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping IDRs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The RECRhlE1-IDRRhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The RECRhlE2-IDRRhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases.

A potential space-making role in cell wall biogenesis for SltB1and DacB revealed by a beta-lactamase induction phenotype in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes the beta-lactamase AmpC, which promotes resistance to beta-lactam antibiotics. Expression of ampC is induced by anhydro-muropeptides (AMPs) released from the peptidoglycan (PG) cell wall upon beta-lactam treatment. AmpC can also be induced via genetic inactivation of PG biogenesis factors such as the endopeptidase DacB that cleaves PG crosslinks. Mutants in dacB occur in beta-lactam-resistant clinical isolates of P. aeruginosa, but it has remained unclear why DacB inactivation promotes ampC induction. Similarly, the inactivation of lytic transglycosylase (LT) enzymes such as SltB1 that cut PG glycans has also been associated with ampC induction and beta-lactam resistance. Given that LT enzymes are capable of producing AMP products that serve as ampC inducers, this latter observation has been especially difficult to explain. Here, we show that ampC induction in sltB1 or dacB mutants requires another LT enzyme called MltG. In Escherichia coli, MltG has been implicated in the degradation of nascent PG strands produced upon beta-lactam treatment. Accordingly, in P. aeruginosa sltB1 and dacB mutants, we detected the MltG-dependent production of pentapeptide-containing AMP products that are signatures of nascent PG degradation. Our results therefore support a model in which SltB1 and DacB use their PG-cleaving activity to open space in the PG matrix for the insertion of new material. Thus, their inactivation mimics low-level beta-lactam treatment by reducing the efficiency of new PG insertion into the wall, causing the degradation of some nascent PG material by MltG to produce the ampC-inducing signal. IMPORTANCE: Inducible beta-lactamases like the ampC system of Pseudomonas aeruginosa are a common determinant of beta-lactam resistance among gram-negative bacteria. The regulation of ampC is elegantly tuned to detect defects in cell wall synthesis caused by beta-lactam drugs. Studies of mutations causing ampC induction in the absence of drug therefore promise to reveal new insights into the process of cell wall biogenesis in addition to aiding our understanding of how resistance to beta-lactam antibiotics arises in the clinic. In this study, the ampC induction phenotype for mutants lacking a glycan-cleaving enzyme or an enzyme that cuts cell wall crosslinks was used to uncover a potential role for these enzymes in making space in the wall matrix for the insertion of new material during cell growth.

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa.

The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.

The detection of KPC-2, NDM-1, and VIM-2 carbapenemases in international clones isolated from fresh vegetables highlights an emerging food safety issue.

Resistance to carbapenems emerged in clinical settings and has rapidly spread to other sectors, such as food and the environment, representing a One Health problem. In this regard, vegetables contaminated by critical priority pathogens have raised global concerns. Here, we have performed a whole-genome sequence-based analysis of extensively drug-resistant Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa strains isolated from cabbage, spinach, and lettuce, respectively. Genomic analysis revealed the emergence of international and high-risk clones belonging to ST340, ST155, and ST233, harboring a broad resistome to clinically important antimicrobials. In this context, K. pneumoniae, E. coli, and P. aeruginosa strains carried blaKPC-2, blaNDM-1, and blaVIM-2, respectively. The blaKPC-2 gene with a non-Tn4401 element (NTEKPC-Ic) was located on an IncX3-IncU plasmid, while the blaVIM-2 gene was associated with a Tn402-like class 1 integron, In559, on the chromosome. Curiously, the blaNDM-1 gene coexisted with the blaPER-2 gene on an IncC plasmid and the regions harboring both genes contained sequences of Tn3-like element ISKox2-like family transposase. Comparative genomic analysis showed interspecies and clonal transmission of carbapenemase-encoding genes at the human-animal-environmental interface. These findings raise a food safety alert about hospital-associated carbapenemase producers, supporting that fresh vegetables can act as a vehicle for the spread of high-risk clones.

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB.

Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli, indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase.IMPORTANCEStenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa. We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

Exploring roles of the chitinase ChiC in modulating Pseudomonas aeruginosa virulence phenotypes.

Chitinases are ubiquitous enzymes involved in biomass degradation and chitin turnover in nature. Pseudomonas aeruginosa (PA), an opportunistic human pathogen, expresses ChiC, a secreted glycoside hydrolase 18 family chitinase. Despite speculation about ChiC's role in PA disease pathogenesis, there is scant evidence supporting this hypothesis. Since PA cannot catabolize chitin, we investigated the potential function(s) of ChiC in PA pathophysiology. Our findings show that ChiC exhibits activity against both insoluble (α- and ß-chitin) and soluble chitooligosaccharides. Enzyme kinetics toward (GlcNAc)4 revealed a kcat of 6.50 s-1 and a KM of 1.38 mM, the latter remarkably high for a canonical chitinase. In our label-free proteomics investigation, ChiC was among the most abundant proteins in the Pel biofilm, suggesting a potential contribution to PA biofilm formation. Using an intratracheal challenge model of PA pneumonia, the chiC::ISphoA/hah transposon insertion mutant paradoxically showed slightly increased virulence compared to the wild-type parent strain. Our results indicate that ChiC is a genuine chitinase that contributes to a PA pathoadaptive pathway.IMPORTANCEIn addition to performing chitin degradation, chitinases from the glycoside hydrolase 18 family have been found to play important roles during pathogenic bacterial infection. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing pneumonia in immunocompromised individuals. Despite not being able to grow on chitin, the bacterium produces a chitinase (ChiC) with hitherto unknown function. This study describes an in-depth characterization of ChiC, focusing on its potential contribution to the bacterium's disease-causing ability. We demonstrate that ChiC can degrade both polymeric chitin and chitooligosaccharides, and proteomic analysis of Pseudomonas aeruginosa biofilm revealed an abundance of ChiC, hinting at a potential role in biofilm formation. Surprisingly, a mutant strain incapable of ChiC production showed higher virulence than the wild-type strain. While ChiC appears to be a genuine chitinase, further investigation is required to fully elucidate its contribution to Pseudomonas aeruginosa virulence, an important task given the evident health risk posed by this bacterium.

(Heteroarylmethyl)benzoic Acids as a New Class of Bacterial Cystathionine γ-Lyase Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling.

Antibiotic resistance is one of the most serious global health threats. Therefore, there is a need to develop antimicrobial agents with new mechanisms of action. Targeting of bacterial cystathionine γ-lyase (bCSE), an enzyme essential for bacterial survival, is a promising approach to overcome antibiotic resistance. Here, we described a series of (heteroarylmethyl)benzoic acid derivatives and evaluated their ability to inhibit bCSE or its human ortholog hCSE using known bCSE inhibitor NL2 as a lead compound. Derivatives bearing the 6-bromoindole group proved to be the most active, with IC50 values in the midmicromolar range, and highly selective for bCSE over hCSE. Furthermore, none of these compounds showed significant toxicity to HEK293T cells. The obtained data were rationalized by ligand-based and structure-based molecular modeling analyses. The most active compounds were also found to be an effective adjunct to several widely used antibacterial agents against clinically relevant antibiotic-resistant strains of such bacteria as Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The most potent compounds, 3h and 3i, also showed a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. Finally, compound 3i manifested potentiating activity in pneumonia, sepsis, and infected-wound in vivo models.

In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa.

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.