UNLABELLED ABSTRACT: BACKGROUND Trichinellosis is a zoonotic disease in humans caused by Trichinella spp. The present study was undertaken to discover excretory-secretory (E-S) proteins from T. spiralis and T. britovi muscle larvae (ML) that hold promise for species-specific diagnostics. To that end, the purified E-S proteins were analyzed by fluorescent two-dimensional difference gel electrophoresis (2-D DIGE) coupled with protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To search for immunoreactive proteins that are specifically recognized by host antibodies the E-S proteins were subjected to two-dimensional (2-DE) immunoblotting with antisera derived from pigs experimentally infected with T. spiralis or T. britovi. RESULTS According to 2-D DIGE analysis, a total of twenty-two proteins including potentially immunogenic proteins and proteins produced only by one of the two Trichinella species were subjected to LC-MS/MS for protein identification. From these proteins seventeen could be identified, of which many were identified in multiple spots, suggesting that they have undergone post-translational modification, possibly involving glycosylation and/or proteolysis. These proteins included 5'-nucleotidase, serine-type protease/proteinase, and p43 glycoprotein (gp43) as well as 49 kDa E-S protein (p49). Our findings also suggest that some of the commonly identified proteins were post-translationally modified to different extents, which in certain cases seemed to result in species-specific modification. Both commonly and specifically recognized immunoreactive proteins were identified by 2-DE immunoblotting; shared antigens were identified as gp43 and different protease variants, whereas those specific to T. britovi included multiple isoforms of the 5'-nucleotidase. CONCLUSIONS Both 2-D DIGE and 2-DE immunoblotting approaches indicate that T. spiralis and T. britovi produce somewhat distinctive antigen profiles, which contain E-S antigens with potential as species-specific diagnostic markers for Trichinella. Our results also demonstrate the value of 2-D DIGE as a versatile tool to compare secretomes of different Trichinella species for pinpointing factors contributing to the interaction with the host.

Acidic environments encountered in food products and during gastrointestinal tract passage affect the survival of bacteria that are marketed as probiotics. In this study, the global proteome responses of the probiotic lactic acid bacterium Lactobacillus rhamnosus GG to two physiologically relevant pH conditions (pH 4.8 and pH 5.8) were studied by 2-D DIGE. The proteomics data were complemented with transcriptome analyses by whole-genome DNA microarrays. The cells were cultured in industrial-type whey medium under a strictly defined bioreactor conditions. In total, 2-D DIGE revealed the pH-dependent formation of 92 protein spots. In response to lower pH conditions, the strongest up-regulation of all proteins was detected for a predicted surface antigen, LGG_02016. In addition, the acid pH was found to up-regulate the expression of F(0)F(1)-ATP synthase genes whereas the abundance of proteins participating in nucleotide biosynthesis and protein synthesis was significantly diminished. Moreover, the results suggest that L. rhamnosus GG modulates its pyruvate metabolism depending on the growth pH. Furthermore, a growth pH-dependent protein phosphorylation phenomenon was detected in several L. rhamnosus GG proteins with ProQ Diamond 2-DE gel staining. Proteins participating in central cellular pathways were shown to be phosphorylated, and the phosphorylation of glycolytic enzymes was found to be especially extensive.

In Staphylococcus aureus, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here reassessed the role of ClpP in SigB-proficient clinical strains. To this end, clpP was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the clpP deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the clpP mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, mgrA, sarZ, sarR, and arlRS was similarly changed in all clpP mutants. ClpP affected the expression of sarS in a strain-dependent manner, and we propose that the differential expression of sarS is central to the strain-dependent effect of ClpP on the expression of virulence genes.

The growth phase during which probiotic bacteria are harvested and consumed can strongly influence their performance as health-promoting agents. In this study, global transcriptomic and proteomic changes were studied in the widely used probiotic Lactobacillus rhamnosus GG during growth in industrial-type whey medium under strictly defined bioreactor conditions. The expression of 636 genes (P ≤ 0.01) and 116 proteins (P < 0.05) changed significantly over time. Of the significantly differentially produced proteins, 61 were associated with alterations at the transcript level. The most remarkable growth phase-dependent changes occurred during the transition from the exponential to the stationary growth phase and were associated with the shift from glucose fermentation to galactose utilization and the transition from homolactic to mixed acid fermentation. Furthermore, several genes encoding proteins proposed to promote the survival and persistence of L. rhamnosus GG in the host and proteins that directly contribute to human health showed temporal changes in expression. Our results suggest that L. rhamnosus GG has a highly flexible and adaptable metabolism and that the growth stage during which bacterial cells are harvested and consumed should be taken into consideration to gain the maximal benefit from probiotic bacteria.

The present study reports an in-depth proteome analysis of two Lactobacillus rhamnosus strains, the well-known probiotic strain GG and the dairy strain Lc705. We used GeLC-MS/MS, in which proteins are separated using 1-DE and identified using nanoLC-MS/MS, to generate high-quality protein catalogs. To maximize the number of identifications, all data sets were searched against the target databases using two search engines, Mascot and Paragon. As a result, over 1600 high-confidence protein identifications, covering nearly 60% of the predicted proteomes, were obtained from each strain. This approach enabled identification of more than 40% of all predicted surfome proteins, including a high number of lipoproteins, integral membrane proteins, peptidoglycan associated proteins, and proteins predicted to be released into the extracellular environment. A comparison of both data sets revealed the expression of more than 90 proteins in GG and 150 in Lc705, which lack evolutionary counterparts in the other strain. Differences were noted in proteins with a likely role in biofilm formation, phage-related functions, reshaping the bacterial cell wall, and immunomodulation. The present study provides the most comprehensive catalog of the Lactobacillus proteins to date and holds great promise for the discovery of novel probiotic effector molecules.

Lactobacillus rhamnosus GG (LGG) is one of the most extensively studied and widely used probiotic bacteria. While the benefits of LGG treatment in gastrointestinal disorders and immunomodulation are well-documented, functional genomics research of this bacterium has only recently been initiated. In the present study, a 2-D DIGE approach was used for the quantitative analysis of growth media-dependent changes in LGG protein abundance. Proteins were isolated from cells grown in industrial-type whey-based medium or in rich laboratory medium for subsequent 2-D DIGE. The analysis revealed patterns of protein abundance unique to each growth condition. In total, 196 quantitatively altered protein spots (at least 1.5-fold change in relative abundance, p < 0.05) representing approximately 13% of all protein spots in the gel were detected. From these protein spots, 157 were identified by mass spectrometry and were found to represent 100 distinct gene products. Collectively, these data show that growth of LGG in whey medium increased the relative abundance of proteins involved in purine biosynthesis, galactose metabolism, and fatty acid biosynthesis. In comparison, growth of LGG in laboratory medium resulted in an increase in the amount of proteins involved in translation and the general stress response, as well as pyrimidine and exopolysaccharide biosynthesis. Moreover, several enzymes of the proteolytic system of LGG demonstrated growth medium-dependent production. The present study demonstrates the fundamental effects of culture conditions on the proteome of LGG, which are likely to affect the functionality and characteristics of its use as a probiotic.

In Streptococcus uberis, the fluoroquinolone antibiotic ciprofloxacin induces a mutagenic response that is distinct from the SOS paradigm. Two-dimensional differential gel electrophoresis was employed to investigate the effect of ciprofloxacin exposure on the proteome of S. uberis. Twenty-four protein spots exhibiting differential expression (p < 0.05) were identified as enzymes with potential role in oxidative stress, NADH generation and nucleotide biosynthesis. We suggest that these metabolic changes provide S. uberis means to stimulate mutagenesis and adaptation.

Streptococcus uberis is an environmental bovine mastitis pathogen capable of UV-inducible SOS mutagenesis. Bacterial SOS systems can be induced by several chemicals including also antibiotics used in clinical practice. Here, we have studied the effect of ciprofloxacin, a fluoroquinolone antibiotic and known inducer of SOS, on mutations leading to antibiotic resistance in S. uberis. Mutation frequencies and spectra were compared in a wild-type S. uberis strain and its DeltaumuC derivative. The results revealed that concentrations of ciprofloxacin corresponding to 0.3-0.5x minimum inhibitory concentration (MIC) induce mutagenesis independent of UmuC. Partial sequencing of the rpoB gene of individual rifampin-resistant clones from wild-type and DeltaumuC strains revealed a similar but complex pattern of point mutations including transitions, transversions and deletions/insertions. It was previously shown that UV induces mainly transition-type mutations and UmuC is essential for the process. Thus, the results presented here demonstrate that S. uberis employs distinct mechanisms for ciprofloxacin and UV-induced mutagenesis, which is a striking difference to Escherichia coli SOS model.

Stress-inducible proteins are likely to contribute to the survival and activity of probiotic bacteria during industrial processes and in the gastrointestinal tract. The recently published genome sequence of probiotic Lactobacillus gasseri ATCC 33323 suggests the presence of ClpC, ClpE, ClpL, and ClpX from the Clp ATPase family of stress proteins. The heat-shock response of L. gasseri was studied using 2-D DIGE. A total of 20 protein spots showing significant (p<0.05) increase in abundance after 30 min heat-shock were identified, including DnaK, GroEL, ClpC, ClpE, and ClpL. To study the physiological role of ClpL, one of the most highly induced proteins during heat-shock, its corresponding gene was inactivated. The DeltaclpL mutant strain had growth characteristics that were indistinguishable from wild-type under several stress conditions. However, in the absence of functional ClpL, L. gasseri exhibited drastically reduced survival at a lethal temperature and was unable to induce thermotolerance. Genome sequences indicate that the expression of clp genes in several Lactobacillus species is regulated by HrcA, instead of CtsR, the conserved clp gene regulator of low G+C Gram-positive bacteria. Electrophoretic mobility shift assays using L. gasseri HrcA protein and clpL upstream fragments revealed, for the first time, a direct interaction between HrcA and the promoter of a clp gene from a Lactobacillus.

Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report identification of a potential self-regulated SOS mutagenesis gene cassette in the Streptococcaceae family. Exposure to UV-light was found to increase mutations to antibiotic resistance in Streptococcus uberis cultures. The mutational spectra revealed mainly G:C-->A:T transitions and Northern analyses demonstrated increased expression of a Y-family DNA polymerase resembling UmuC under DNA-damaging conditions. In the absence of the Y-family polymerase, S. uberis cells were sensitive to UV-light and to mitomycin C. Furthermore, the UV-induced mutagenesis was almost completely abolished in cells deficient in the Y-family polymerase. The gene encoding the the Y-family polymerase was localized in a four gene operon including two hypothetical genes and a gene encoding a HdiR homolog. Electrophoretic mobility shift assays demonstrated that S. uberis HdiR binds specifically to an inverted repeat sequence in the promoter region of the four gene operon. Data base searches revealed conservation of the gene cassette in several Streptococcus species including at least one genome of Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Streptococcus sanguinis, and Streptococcus thermophilus strains. In addition, the umuC-operon was localized in several mobile DNA elements of Streptococcus and Lactococcus species. We conclude that the hdiR-umuC-ORF3-ORF4 operon represents a novel gene cassette capable of mediating of SOS mutagenesis among members of Streptococcaceae.