Stress Physiology of Lactic Acid Bacteria Papadimitriou, Konstantinos; Alegría, Ángel; Bron, Peter A ...
Microbiology and molecular biology reviews,
09/2016, Letnik:
80, Številka:
3
Journal Article
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Lactic acid bacteria (LAB) are important starter, commensal, or pathogenic microorganisms. The stress physiology of LAB has been studied in depth for over 2 decades, fueled mostly by the ...technological implications of LAB robustness in the food industry. Survival of probiotic LAB in the host and the potential relatedness of LAB virulence to their stress resilience have intensified interest in the field. Thus, a wealth of information concerning stress responses exists today for strains as diverse as starter (e.g., Lactococcus lactis), probiotic (e.g., several Lactobacillus spp.), and pathogenic (e.g., Enterococcus and Streptococcus spp.) LAB. Here we present the state of the art for LAB stress behavior. We describe the multitude of stresses that LAB are confronted with, and we present the experimental context used to study the stress responses of LAB, focusing on adaptation, habituation, and cross-protection as well as on self-induced multistress resistance in stationary phase, biofilms, and dormancy. We also consider stress responses at the population and single-cell levels. Subsequently, we concentrate on the stress defense mechanisms that have been reported to date, grouping them according to their direct participation in preserving cell energy, defending macromolecules, and protecting the cell envelope. Stress-induced responses of probiotic LAB and commensal/pathogenic LAB are highlighted separately due to the complexity of the peculiar multistress conditions to which these bacteria are subjected in their hosts. Induction of prophages under environmental stresses is then discussed. Finally, we present systems-based strategies to characterize the "stressome" of LAB and to engineer new food-related and probiotic LAB with improved stress tolerance.
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.
The present study investigated the effect of co-fermentation on vitamin B12 content and microbiological composition of wheat bran.
DSM 20271 was used as the producer of vitamin while
ATCC 14869 was ...selected to ensure the microbial safety of the bran dough. Fermentation trials were conducted in bioreactors to monitor and adjust the pH of the ferments. Vitamin B12 level reached 357 ± 8 ng/g dry weight (dw) after 1 day of pH-controlled fermentation with
monoculture and remained stable thereafter. In co-fermentation with
, slightly less vitamin B12 (255 ± 31 ng/g dw) was produced in 1 day and an effective inhibition of the growth of total
and
was obtained. On day 3, vitamin B12 content in pH-controlled co-fermentation increased to 332 ± 44 ng/g dw. On the other hand, without a pH control, co-fermentation resulted in a stronger inhibition of
and
but a lower level of vitamin B12 (183 ± 5 ng/g dw on day 3). These results demonstrated that wheat bran fermented by
and
can be a promising way to produce vitamin B12 fortified plant-origin food ingredients, which could reduce cereal waste streams and contribute to a more resilient food chain.
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.
Propionibacterium freudenreichii is an industrially important bacterium granted the Generally Recognized as Safe (the GRAS) status, due to its long safe use in food bioprocesses. Despite the ...recognized role in the food industry and in the production of vitamin B12, as well as its documented health-promoting potential, P. freudenreichii remained poorly characterised at the genomic level. At present, only three complete genome sequences are available for the species.
We used the PacBio RS II sequencing platform to generate complete genomes of 20 P. freudenreichii strains and compared them in detail. Comparative analyses revealed both sequence conservation and genome organisational diversity among the strains. Assembly from long reads resulted in the discovery of additional circular elements: two putative conjugative plasmids and three active, lysogenic bacteriophages. It also permitted characterisation of the CRISPR-Cas systems. The use of the PacBio sequencing platform allowed identification of DNA modifications, which in turn allowed characterisation of the restriction-modification systems together with their recognition motifs. The observed genomic differences suggested strain variation in surface piliation and specific mucus binding, which were validated by experimental studies. The phenotypic characterisation displayed large diversity between the strains in ability to utilise a range of carbohydrates, to grow at unfavourable conditions and to form a biofilm.
The complete genome sequencing allowed detailed characterisation of the industrially important species, P. freudenreichii by facilitating the discovery of previously unknown features. The results presented here lay a solid foundation for future genetic and functional genomic investigations of this actinobacterial species.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Copper shows a high promise in developing biomedical materials with antibacterial effect. The antibacterial effect can be enhanced by nanostructured surfaces with superhydrophobic properties, which ...reduce the solid contact area available for bacterial adhesion and adherent growth. Here, three structured surfaces are fabricated to test the combined effect of copper and superhydrophobicity for antibacterial effects. One of the samples is superhydrophobic but does not contain copper, one contains copper but is not superhydrophobic, and the third is both superhydrophobic and contained copper. The antibiofilm and bactericidal effects of these samples are tested against medically important Gram‐positive and ‐negative bacteria including Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), and Pseudomonas aeruginosa (P. aeruginosa). The findings indicate that copper alone without superhydrophobicity, while decreasing the cell viability in most of the tested species, supports remarkably more biomass compared to the reference sample. The superhydrophobic and copper bearing samples, while allowing adherent growth to take place, provide the greatest bactericidal effect against two P. aeruginosa strains, and both the antibiofilm and/or bactericidal effects against S. aureus and S. epidermidis. Thus, this study reports that nanostructured materials, combining superhydrophobicity with copper, can be the method of choice to neutralize pathogens with different cell‐wall structures and surface components mediating adherent growth.
It is shown how two separate antibacterial effects can be combined on the same surface: the element copper and superhydrophobicity. These two properties are studied separately and combined with micro/nanostructured and copper‐containing and non‐copper‐containing surfaces. The biofilm tests use four clinically relevant biofilm‐forming bacterial species. The best antibacterial performance is obtained on the surface, which combines both properties.
Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and a promising target for the development of vaccines and antimicrobial compounds against Staphylococcus aureus ...Here we demonstrate that mutations in the conditionally essential ltaS (LTA synthase) gene arise spontaneously in an S. aureus mutant lacking the ClpX chaperone. A wide variety of ltaS mutations were selected, and among these, a substantial portion resulted in premature stop codons and other changes predicted to abolish LtaS synthesis. Consistent with this assumption, the clpX ltaS double mutants did not produce LTA, and genetic analyses confirmed that LTA becomes nonessential in the absence of the ClpX chaperone. In fact, inactivation of ltaS alleviated the severe growth defect conferred by the clpX deletion. Microscopic analyses showed that the absence of ClpX partly alleviates the septum placement defects of an LTA-depleted strain, while other phenotypes typical of LTA-negative S. aureus mutants, including increased cell size and decreased autolytic activity, are retained. In conclusion, our results indicate that LTA has an essential role in septum placement that can be bypassed by inactivating the ClpX chaperone.
Lipoteichoic acid is an essential component of the Staphylococcus aureus cell envelope and an attractive target for the development of vaccines and antimicrobials directed against antibiotic-resistant Gram-positive bacteria such as methicillin-resistant S. aureus and vancomycin-resistant enterococci. In this study, we showed that the lipoteichoic acid polymer is essential for growth of S. aureus only as long as the ClpX chaperone is present in the cell. Our results indicate that lipoteichoic acid and ClpX play opposite roles in a pathway that controls two key cell division processes in S. aureus, namely, septum formation and autolytic activity. The discovery of a novel functional connection in the genetic network that controls cell division in S. aureus may expand the repertoire of possible strategies to identify compounds or compound combinations that kill antibiotic-resistant S. aureus.
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