Bacterial Two Component Systems: Overexpression and Purification: In Vitro and In Vivo Inhibitor Screens.

Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy, such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses upon cell wall or cell membrane damage. However, (i) activity assays using truncated kinase proteins, that is, the cytoplasmic domains containing the conserved histidine residue for phosphorylation, have been shown to produce artifacts, and (ii) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from Gram-positive bacteria that do not harbor more than two trans-membrane domains in an Escherichia coli host. This guide also presents in vitro and in vivo phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases, either using a traditional radioactively labeled ATP assay to quantify histidine kinase phosphorylation or Phos-tag acrylamide gel electrophoresis to examine histidine kinase phosphorylation through mobility shift in the polyacrylamide gel. In addition, we describe the use of Phos-tag combined with a western blot approach to visualize the phosphorylation of a response regulator in vivo.

Cervimycin-Resistant Staphylococcus aureus Strains Display Vancomycin-Intermediate Resistant Phenotypes.

Resistance to antibiotics is an increasing problem and necessitates novel antibacterial therapies. The polyketide antibiotics cervimycin A to D are natural products of Streptomyces tendae HKI 0179 with promising activity against multidrug-resistant staphylococci and vancomycin-resistant enterococci. To initiate mode of action studies, we selected cervimycin C- and D-resistant (CmR) Staphylococcus aureus strains. Genome sequencing of CmR mutants revealed amino acid exchanges in the essential histidine kinase WalK, the Clp protease proteolytic subunit ClpP or the Clp ATPase ClpC, and the heat shock protein DnaK. Interestingly, all characterized CmR mutants harbored a combination of mutations in walK and clpP or clpC. In vitro and in vivo analyses showed that the mutations in the Clp proteins abolished ClpP or ClpC activity, and the deletion of clpP rendered S. aureus but not all Bacillus subtilis strains cervimycin-resistant. The essential gene walK was the second mutational hotspot in the CmR S. aureus strains, which decreased WalK activity in vitro and generated a vancomycin-intermediate resistant phenotype, with a thickened cell wall, a lower growth rate, and reduced cell lysis. Transcriptomic and proteomic analyses revealed massive alterations in the CmR strains compared to the parent strain S. aureus SG511, with major shifts in the heat shock regulon, the metal ion homeostasis, and the carbohydrate metabolism. Taken together, mutations in the heat shock genes clpP, clpC, and dnaK, and the walK kinase gene in CmR mutants induced a vancomycin-intermediate resistant phenotype in S. aureus, suggesting cell wall metabolism or the Clp protease system as primary target of cervimycin. IMPORTANCE Staphylococcus aureus is a frequent cause of infections in both the community and hospital setting. Resistance development of S. aureus to various antibiotics is a severe problem for the treatment of this pathogen worldwide. New powerful antimicrobial agents against Gram-positives are needed, since antibiotics like vancomycin fail to cure vancomycin-intermediate resistant S. aureus (VISA) and vancomycin-resistant enterococci (VRE) infections. One candidate substance with promising activity against these organisms is cervimycin, which is an antibiotic complex with a yet unknown mode of action. In our study, we provide first insights into the mode of action of cervimycins. By characterizing cervimycin-resistant S. aureus strains, we revealed the Clp system and the essential kinase WalK as mutational hotspots for cervimycin resistance in S. aureus. It further emerged that cervimycin-resistant S. aureus strains show a VISA phenotype, indicating a role of cervimycin in perturbing the bacterial cell envelope.

Bacteria isolated from hospital, municipal and slaughterhouse wastewaters show characteristic, different resistance profiles.

Multidrug-resistant bacteria cause difficult-to-treat infections and pose a risk for modern medicine. Sources of multidrug-resistant bacteria include hospital, municipal and slaughterhouse wastewaters. In this study, bacteria with resistance to 3rd generation cephalosporins were isolated from all three wastewater biotopes, including a maximum care hospital, municipal wastewaters collected separately from a city and small rural towns and the wastewaters of two pig and two poultry slaughterhouses. The resistance profiles of all isolates against clinically relevant antibiotics (including ß-lactams like carbapenems, the quinolone ciprofloxacin, colistin, and trimethoprim/sulfamethoxazole) were determined at the same laboratory. The bacteria were classified according to their risk to human health using clinical criteria, with an emphasis on producers of carbapenemases, since carbapenems are prescribed for hospitalized patients with infections with multi-drug resistant bacteria. The results showed that bacteria that pose the highest risk, i. e., bacteria resistant to all ß-lactams including carbapenems and ciprofloxacin, were mainly disseminated by hospitals and were present only in low amounts in municipal wastewater. The isolates from hospital wastewater also showed the highest rates of resistance against antibiotics used for treatment of carbapenemase producers and some isolates were susceptible to only one antibiotic substance. In accordance with these results, qPCR of resistance genes showed that 90% of the daily load of carbapenemase genes entering the municipal wastewater treatment plant was supplied by the clinically influenced wastewater, which constituted approximately 6% of the wastewater at this sampling point. Likewise, the signature of the clinical wastewater was still visible in the resistance profiles of the bacteria isolated at the entry into the wastewater treatment plant. Carbapenemase producers were not detected in slaughterhouse wastewater, but strains harboring the colistin resistance gene mcr-1 could be isolated. Resistances against orally available antibiotics like ciprofloxacin and trimethoprim/sulfamethoxazole were widespread in strains from all three wastewaters.

YycH and YycI Regulate Expression of Staphylococcus aureus Autolysins by Activation of WalRK Phosphorylation.

Staphylococcus aureus is a facultative pathogen that can encode numerous antibiotic resistance and immune evasion genes and can cause severe infections. Reduced susceptibility to last resort antibiotics such as vancomycin and daptomycin is often associated with mutations in walRK, an essential two-component regulatory system (TCS). This study focuses on the WalK accessory membrane proteins YycH and YycI and their influence on WalRK phosphorylation. Depletion of YycH and YycI by antisense RNA caused an impaired autolysis, indicating a positive regulatory function on WalK as has been previously described. Phosphorylation assays with full-length recombinant proteins in phospholipid liposomes showed that YycH and YycI stimulate WalK activity and that both regulatory proteins are needed for full activation of the WalK kinase. This was validated in vivo through examining the phosphorylation status of WalR using Phos-tag SDS-PAGE with a yycHI deletion mutant exhibiting reduced levels of phosphorylated WalR. In the yycHI knockdown strain, muropeptide composition of the cell wall was not affected, however, the wall teichoic acid content was increased. In conclusion, a direct modulation of WalRK phosphorylation activity by the accessory proteins YycH and YycI is reported both in vitro and in vivo. Taken together, our results show that YycH and YycI are important in the direct regulation of WalRK-dependent cell wall metabolism.

Zinc-binding to the cytoplasmic PAS domain regulates the essential WalK histidine kinase of Staphylococcus aureus.

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introducing the WalKH271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PASCYT domain reveal a metal-binding site, in which a zinc ion (Zn2+) is tetrahedrally-coordinated by four amino acids including H271. The WalKH271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon.

Detection of methicillin-resistant coagulase-negative staphylococci harboring the class A mec complex by MALDI-TOF mass spectrometry.

The aim of this study was to test the identification of methicillin resistance in coagulase-negative staphylococci by routine matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). SCCmec cassettes of type II, III and VIII encode a small peptide called PSM-mec in the vicinity of mecA. It is visible at m/z 2415 during MALDI-TOF MS of whole cells of Staphylococcus aureus. In view of the fact that psm-mec has been identified in methicillin-resistant coagulase-negative staphylococci, we evaluated a collection of clinical coagulase-negative staphylococci, that contained 77.03% of methicillin-resistant isolates, for the presence of the structural gene encoding PSM-mec and the appearance of the corresponding signal during mass spectroscopy. In MALDI-TOF MS spectra, 89.65% of the strains that harbored the gene yielded the correct signal, corresponding to a sensitivity of 0.897 and a specificity of 1.0. However, regarding detection of methicillin resistance, i. e. considering all resistant strains as positive regardless of the presence of the gene, the overall sensitivity of the test decreased to 0.285, due to the fact that only 29.43% of all resistant isolates contained psm-mec. In conclusion, the presence of the signal in MALDI-TOF MS quickly indicates methicillin-resistance in coagulase-negative staphylococci but its absence does not indicate susceptibility to methicillin.

Dissemination of multi-resistant Gram-negative bacteria into German wastewater and surface waters.

Carbapenem antibiotics constitute the mainstay therapy of nosocomial infections with extended spectrum beta-lactamase producing Gram-negative bacteria; however, resistance against these compounds is increasing. This study was designed to demonstrate that carbapenemase-producing bacteria are disseminated from hospitals into the environment. To this end, resistant bacteria were isolated from a clinical/urban and from a rural catchment system in Germany in 2016/17. The study followed the dissemination of resistant bacteria from the wastewater through the wastewater treatment plant (WWTP) into the receiving surface waters. The bacteria were cultivated on selective agar and characterized by antibiotic testing, real-time PCR targeting carbapenemase genes and typing. Bacteria with resistance to third generation cephalosporins were isolated from all sample sites. 134 isolates harboring carbapenemase genes encoding VIM, NDM and OXA-48 and 26 XDR (extensively drug-resistant) strains with susceptibility to only one or two antibiotics were isolated from the clinical/urban system. The rural system yielded eight carbapenemase producers and no XDR strains. In conclusion, clinical wastewaters were charged with a high proportion of multidrug resistant bacteria. Although most of these bacteria were eliminated during wastewater treatment, dissemination into surface waters is possible as single carbapenemase producers were still present in the effluent of the WWTP.

Nasal Carriage of Staphylococcus aureus among Children in the Ashanti Region of Ghana.

BACKGROUND: Nasal carriage with Staphylococcus aureus is a common risk factor for invasive infections, indicating the necessity to monitor prevalent strains, particularly in the vulnerable paediatric population. This surveillance study aims to identify carriage rates, subtypes, antimicrobial susceptibilities and virulence markers of nasal S. aureus isolates collected from children living in the Ashanti region of Ghana. METHODS: Nasal swabs were obtained from children < 15 years of age on admission to the Agogo Presbyterian Hospital between April 2014 and January 2015. S. aureus isolates were characterized by their antimicrobial susceptibility, the presence of genes encoding for Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1) and further differentiated by spa-typing and multi-locus-sequence-typing. RESULTS: Out of 544 children 120 (22.1%) were colonized with S. aureus, with highest carriage rates during the rainy seasons (27.2%; p = 0.007), in females aged 6-8 years (43.7%) and males aged 8-10 years (35.2%). The 123 isolates belonged to 35 different spa-types and 19 sequence types (ST) with the three most prevalent spa-types being t355 (n = 25), t84 (n = 18), t939 (n = 13), corresponding to ST152, ST15 and ST45. Two (2%) isolates were methicillin-resistant S. aureus (MRSA), classified as t1096 (ST152) and t4454 (ST45), and 16 (13%) were resistant to three or more different antimicrobial classes. PVL and TSST-1 were detected in 71 (58%) and 17 (14%) isolates respectively. CONCLUSION: S. aureus carriage among Ghanaian children seems to depend on age, sex and seasonality. While MRSA rates are low, the high prevalence of PVL is of serious concern as these strains might serve not only as a source for severe invasive infections but may also transfer genes, leading to highly virulent MRSA clones.

Differentiation of Staphylococcus argenteus (formerly: Staphylococcus aureus clonal complex 75) by mass spectrometry from S. aureus using the first strain isolated from a wild African great ape.

The species Staphylococcus argenteus was separated recently from Staphylococcus aureus (Tong S.Y., F. Schaumburg, M.J. Ellington, J. Corander, B. Pichon, F. Leendertz, S.D. Bentley, J. Parkhill, D.C. Holt, G. Peters, and P.M. Giffard, 2015). The objective of this work was to characterise the genome of a non-human S. argenteus strain, which had been isolated from the faeces of a wild-living western lowland gorilla in Gabon, and analyse the spectrum of this species in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The full genome sequence revealed a scarcity of virulence genes and absence of resistance genes, indicating a decreased virulence potential compared to S. aureus and the human methicillin-resistant S. argenteus isolate MSHR1132T. Spectra obtained by MALDI-TOF MS and the analysis of available sequences in the genome databases identified several MALDI-TOF MS signals that clearly differentiate S. argenteus, the closely related Staphylococcus schweitzeri and S. aureus. In conclusion, in the absence of biochemical tests that identify the three species, mass spectrometry should be employed as method of choice.

The cell wall precursor lipid II acts as a molecular signal for the Ser/Thr kinase PknB of Staphylococcus aureus.

The assembly of the bacterial cell wall requires synchronization of a multitude of biosynthetic machineries and regulatory networks. The eukaryotic-like serine/threonine kinase PknB has been implicated in coordinating cross-wall formation, autolysis and cell division in Staphylococcus aureus. However, the signal molecule sensed by this kinase remained elusive so far. Here, we provide compelling biochemical evidence that PknB interacts with the ultimate cell wall precursor lipid II, triggering kinase activity. Moreover, we observed crosstalk of PknB with the two component system WalKR and identified the early cell division protein FtsZ as another PknB phosphorylation substrate in S. aureus. In agreement with the implied role in regulation of cell envelope metabolism, we found PknB to preferentially localize to the septum of S. aureus and the PASTA domains to be crucial for recruitment to this site. The data provide a model for the contribution of PknB to control cell wall metabolism and cell division.

Bacterial Histidine Kinases: Overexpression, Purification, and Inhibitor Screen.

Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses like the cell wall stress response. However, (1) activity assays using the truncated phosphorylation domains have been shown to produce artifacts and (2) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from gram-positive bacteria that do not harbor more than two trans-membrane domains using an Escherichia coli host. This guide also presents in vitro phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases using radioactively labeled ATP and, as a novel approach, Phos-tag acrylamide gel electrophoresis to detect phosphorylated proteins by mobility shift in the polyacrylamide gel.