Clp proteolytic complexes consisting of a proteolytic core flanked by Clp ATPases are widely conserved in bacteria, and their biological roles have received considerable interest. In particular, ...mutants in the clp genes in the low-GC-content Gram-positive phyla Bacillales and Lactobacillales display a diverse range of phenotypic changes including general stress sensitivity, aberrant cell morphology, failure to initiate developmental programs, and for pathogens, severely attenuated virulence. Extensive research dedicated to unravelling the molecular mechanisms underlying these complex phenotypes has led to fascinating new insights that will be covered by this review. First, Clp ATPases and ClpP-containing proteolytic complexes play indispensable roles in cellular protein quality control systems by refolding or degrading damaged proteins in both stressed and non-stressed cells. Secondly, ClpP proteases and the chaperone activity of Clp ATPases are important for controlling stability and activity of central transcriptional regulators, thereby exerting tremendous impact on cell physiology. Targets include major stress regulators like Spx (oxidative stress), the antisigma factor RsiW (alkaline stress) and HdiR (DNA damage) in addition to regulators of developmental programs like ComK (competence development), σH and Sda (sporulation). Thus, Clp proteins are central in co-ordinating developmental decisions and stress response in low GC Gram-positive bacteria.
In recent years, bacterial infections have become a main concern following the spread of antimicrobial resistance. In addition, bacterial biofilms are known for their high tolerance to antimicrobials ...and they are regarded as a main cause of recalcitrant infections in humans. Many efforts have been deployed in order to find new antibacterial therapeutic options and the high-throughput screening (HTS) of large libraries of compounds is one of the utilized strategies. However, HTS efforts for anti-biofilm discovery remain uncommon. Here, we miniaturized a 96-well plate (96WP) screening platform, into a 384-well plate (384WP) format, based on a sequential viability and biomass measurements for the assessment of anti-biofilm activity. During the assay optimization process, different parameters were evaluated while using
and
as the bacterial models. We compared the performance of the optimized 384WP platform to our previously established 96WP-based platform by carrying out a pilot screening of 100 compounds, followed by the screening of a library of 2000 compounds to identify new repurposed anti-biofilm agents. Our results show that the optimized 384WP platform is well-suited for screening purposes, allowing for the rapid screening of a higher number of compounds in a run in a reliable manner.
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•Choice of the biofilm formation assay is crucial when designing functional surfaces.•Use of multiple surface parameters enables comprehensive bacteria-surface analysis.•Several ...topographical parameters correlate to phenotypic responses of S. aureus.•Abundance profiles of several virulence proteins correlate to roughness parameters.
Investigating and understanding the response of microbes to various surfaces requires a versatile parametrisation of the surface, and multiple assays that captures the complexity of the biofilm structures. Here, Staphylococcus aureus biofilm viability, polysaccharide poly-N-acetylglucosamine, and proteins on the cell surface were analysed with agar plate- and well plate-based biofilm formation assays. Biofilms were grown on a set of nanostructured polymeric surfaces, which were thoroughly characterised for their surface chemistry and topography. Surface hydrophobicity, summit density as well as peak and valley structure were found to influence the microbial viability and exopolysaccharide abundance level in the agar plate assay. In the well plate assay, surface chemical parameters had a lesser influence on the viability, but roughness caused by valley structures increased the viability and decreased the exopolysaccharide expression. Surface proteins relating to pathogenicity were affected by the biofilm formation assay. The abundance profile of these proteins correlated clearly with several roughness parameters, especially fine structure parameters in the agar plate assay and lateral roughness in the well plate assay. These results highlight the necessity of describing the material surfaces with a versatile set of different roughness parameters to completely understand what specific features of a surface drive a certain bacterial response.
Inhibiting quorum sensing (QS), a central communication system, is a promising strategy to combat bacterial pathogens without antibiotics. Here, we designed novel hybrid compounds targeting the PQS (
...quinolone signal)-dependent quorum sensing (QS) of
that is one of the multidrug-resistant and highly virulent pathogens with urgent need of new antibacterial strategies. We synthesized 12 compounds using standard procedures to combine halogen-substituted anthranilic acids with 4-(2-aminoethyl/4-aminobuthyl)amino-7-chloroquinoline, linked via 1,3,4-oxadiazole. Their antibiofilm activities were first pre-screened using Gram-negative
-based reporter, which identified compounds
-
and
with the highest anti-QS and minimal bactericidal effects in a single experiment. These five compounds were then evaluated against
PAO1 to assess their ability to prevent biofilm formation, eradicate pre-formed biofilms, and inhibit virulence using pyocyanin as a representative marker. Compound
displayed the most potent antibiofilm effect, reducing biofilm formation by nearly 50% and pre-formed biofilm masses by 25%. On the other hand, compound
exhibited the most significant antivirulence effect, reducing pyocyanin synthesis by over 70%. Thus, our study highlights the potential of 1,3,4-oxadiazoles
and
as promising scaffolds to combat
. Additionally, interactive QS systems should be considered to achieve maximal anti-QS activity against this clinically relevant species.
Lactobacillus rhamnosus GG (GG) is a widely used and intensively studied probiotic bacterium. Although the health benefits of strain GG are well documented, the systematic exploration of mechanisms ...by which this strain exerts probiotic effects in the host has only recently been initiated. The ability to survive the harsh conditions of the gastrointestinal tract, including gastric juice containing bile salts, is one of the vital characteristics that enables a probiotic bacterium to transiently colonize the host. Here we used gene expression profiling at the transcriptome and proteome levels to investigate the cellular response of strain GG toward bile under defined bioreactor conditions. The analyses revealed that in response to growth of strain GG in the presence of 0.2% ox gall the transcript levels of 316 genes changed significantly (p < 0.01, t test), and 42 proteins, including both intracellular and surface-exposed proteins (i.e. surfome), were differentially abundant (p < 0.01, t test in total proteome analysis; p < 0.05, t test in surfome analysis). Protein abundance changes correlated with transcriptome level changes for 14 of these proteins. The identified proteins suggest diverse and specific changes in general stress responses as well as in cell envelope-related functions, including in pathways affecting fatty acid composition, cell surface charge, and thickness of the exopolysaccharide layer. These changes are likely to strengthen the cell envelope against bile-induced stress and signal the GG cells of gut entrance. Notably, the surfome analyses demonstrated significant reduction in the abundance of a protein catalyzing the synthesis of exopolysaccharides, whereas a protein dedicated for active removal of bile compounds from the cells was up-regulated. These findings suggest a role for these proteins in facilitating the well founded interaction of strain GG with the host mucus in the presence of sublethal doses of bile. The significance of these findings in terms of the functionality of a probiotic bacterium is discussed.
Bacterial biofilms have clear implications in disease and in food applications involving probiotics. Here, we show that switching the carbohydrate source from glucose to fructose increased the ...biofilm formation and the total surface-antigenicity of a well-known probiotic,
GG. Surfaceomes (all cell surface-associated proteins) of GG cells grown with glucose and fructose in planktonic and biofilm cultures were identified and compared, which indicated carbohydrate source-dependent variations, especially during biofilm growth. The most distinctive differences under these conditions were detected with several surface adhesins (e.g., MBF, SpaC pilus protein and penicillin-binding proteins), enzymes (glycoside hydrolases, PrsA, PrtP, PrtR, and HtrA) and moonlighting proteins (glycolytic, transcription/translation and stress-associated proteins, r-proteins, tRNA synthetases, Clp family proteins, PepC, PepN, and PepA). The abundance of several known adhesins and candidate moonlighters, including enzymes acting on casein-derived peptides (ClpP, PepC, and PepN), increased in the biofilm cells grown on fructose, from which the surface-associated aminopeptidase activity mediated by PepC and PepN was further confirmed by an enzymatic assay. The mucus binding factor (MBF) was found most abundant in fructose grown biofilm cells whereas SpaC adhesin was identified specifically from planktonic cells growing on fructose. An additional indirect ELISA indicated both growth mode- and carbohydrate-dependent differences in abundance of SpaC, whereas the overall adherence of GG assessed with porcine mucus indicated that the carbon source and the growth mode affected mucus adhesion. The adherence of GG cells to mucus was almost completely inhibited by anti-SpaC antibodies regardless of growth mode and/or carbohydrate source, indicating the key role of the SpaCBA pilus in adherence under the tested conditions. Altogether, our results suggest that carbon source and growth mode coordinate mechanisms shaping the proteinaceous composition of GG cell surface, which potentially contributes to resistance, nutrient acquisition and cell-cell interactions under different conditions. In conclusion, the present study shows that different growth regimes and conditions can have a profound impact on the adherent and antigenic features of GG, thereby providing new information on how to gain additional benefits from this probiotic.
Medical device-associated staphylococcal infections are a common and challenging problem. However, detailed knowledge of staphylococcal biofilm dynamics on clinically relevant surfaces is still ...limited. In the present study, biofilm formation of the
ATCC 25923 strain was studied on clinically relevant materials-borosilicate glass, plexiglass, hydroxyapatite, titanium and polystyrene-at 18, 42 and 66 h. Materials with the highest surface roughness and porosity (hydroxyapatite and plexiglass) did not promote biofilm formation as efficiently as some other selected materials. Matrix-associated poly-
-acetyl-β-(1-6)-glucosamine (PNAG) was considered important in young (18 h) biofilms, whereas proteins appeared to play a more important role at later stages of biofilm development. A total of 460 proteins were identified from biofilm matrices formed on the indicated materials and time points-from which, 66 proteins were proposed to form the core surfaceome. At 18 h, the appearance of several r-proteins and glycolytic adhesive moonlighters, possibly via an autolysin (AtlA)-mediated release, was demonstrated in all materials, whereas classical surface adhesins, resistance- and virulence-associated proteins displayed greater variation in their abundances depending on the used material. Hydroxyapatite-associated biofilms were more susceptible to antibiotics than biofilms formed on titanium, but no clear correlation between the tolerance and biofilm age was observed. Thus, other factors, possibly the adhesive moonlighters, could have contributed to the observed chemotolerant phenotype. In addition, a protein-dependent matrix network was observed to be already well-established at the 18 h time point. To the best of our knowledge, this is among the first studies shedding light into matrix-associated surfaceomes of S. aureus biofilms grown on different clinically relevant materials and at different time points.
Mycobacterium marinum, a slow-growing Actinobacterium, typically induces tuberculosis-like disease in fish. Here, we report a new reference sequence for M. marinum ATCC 927T, along with its DNA ...methylome. This aims to maximize the research potential of this type strain and facilitates investigations into the pathomechanisms of human tuberculosis.
In an effort to find new repurposed antibacterial compounds, we performed the screening of an FDA-approved compounds library against Staphylococcus aureus American Type Culture Collection (ATCC) ...25923. Compounds were evaluated for their capacity to prevent both planktonic growth and biofilm formation as well as to disrupt pre-formed biofilms. One of the identified initial hits was fingolimod (FTY720), an immunomodulator approved for the treatment of multiple sclerosis, which was then selected for follow-up studies. Fingolimod displayed a potent activity against S. aureus and S. epidermidis with a minimum inhibitory concentration (MIC) within the range of 12–15 µM at which concentration killing of all the bacteria was confirmed. A time–kill kinetic study revealed that fingolimod started to drastically reduce the viable bacterial count within two hours and we showed that no resistance developed against this compound for up to 20 days. Fingolimod also displayed a high activity against Acinetobacter baumannii (MIC 25 µM) as well as a modest activity against Escherichia coli and Pseudomonas aeruginosa. In addition, fingolimod inhibited quorum sensing in Chromobacterium violaceum and might therefore target this signaling pathway in certain Gram-negative bacteria. In conclusion, we present the identification of fingolimod from a compound library and its evaluation as a potential repurposed antibacterial compound.
Anoplocephala perfoliata
is a common tapeworm in horses causing colic and even mortalities. Current diagnostic tests to detect
A. perfoliata
infections have their limitations and an improved method ...is needed. Immunoreactive excretory/secretory proteins (E/S proteome) of this parasite can provide promising candidates for diagnostic tests. We compared E/S proteins produced by small (length < 20 mm, width < 5 mm) and large (length 20 to 40 mm, width 5 to 10 mm)
A. perfoliata
worms
in vitro
by label-free quantitative proteomics using a database composed of related
Hymenolepis diminuta, Echinococcus multilocularis/granulosus
and
Taenia aseatica
proteins for protein identifications. Altogether, 509 E/S proteins were identified after incubating the worms
in vitro
for three and eight hours. The greatest E/S proteome changes suggested both worm size- and time-dependent changes in cytoskeleton remodeling, apoptosis, and production of antigens/immunogens. The E/S proteins collected at the three-hour time point represented the natural conditions better than those collected at the eight-hour time point, and thereby contained the most relevant diagnostic targets. Immunoblotting using antibodies from horses tested positive/negative for
A. perfoliata
indicated strongest antigenicity/immunogenicity with 13-, 30- and 100-kDa proteins, involving a thioredoxin, heat-shock chaperone 90 (Hsp90), dynein light chain component (DYNLL), tubulin-specific chaperone A (TBCA) and signaling pathway modulators (14-3-3 and Sj-Ts4). This is among the first studies identifying new diagnostic targets and
A. perfoliata
antigens eliciting a IgG-response in horses.
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