The Exoproteome of Staphylococcus pasteuri Isolated from Cervical Mucus during the Estrus Phase in Water Buffalo (Bubalus bubalis)

Abstract

Bacterial extracellular proteins participate in the host cell communication by virtue of the modulation of pathogenicity, commensalism and mutualism. Studies on the microbiome of cervical mucus of the water buffalo (Bubalus bubalis) have shown the occurrence of Staphylococcus pasteuri and that the presence of this bacterium is indicative of various physiological and reproductive states in the host. Recently, S. pasteuri has been isolated from the cervical mucus of the buffalo during the different phases of estrous cycle, and has proved to be much more pronounced during the estrus phase. The basis underlying the availability of a significantly increased S. pasteuri population, specifically during the estrus phase, is not known. Consequently, it is important to determine the significance of the specific abundance of S. pasteuri during the estrus phase of the buffalo host, particularly from the perspective of whether this bacterial species is capable of contributing to sexual communication via its extracellular proteins and volatiles. Therefore, the relevance of S. pasteuri exoproteome in the buffalo cervical mucus during the estrus phase was analyzed using LC-MS/MS. As many as 219 proteins were identified, among which elongation factor Tu (EF-Tu), 60-kDa chaperonin (Cpn60), enolase, fructose-bisphosphate aldolase class 1 (FBP aldolase), enoyl-[acyl-carrier-protein] reductase [NADPH] (ENR) and lipoprotein (Lpp) were the functionally important candidates. Most of the proteins present in the exoproteome of S. pasteuri were those involved in cellular-metabolic functions, as well as catalytic- and binding activities. Moreover, computational studies of Lpp have shown enhanced interaction with volatiles such as acetic-, butanoic-, isovaleric- and valeric acids, which were identified in the cervical mucus S. pasteuri culture supernatant. The present findings suggest that S. pasteuri extracellular proteins may play an important role in buffalo sexual communication during the estrus phase.

Keywords: chemical communication; cpn60; enolase; extracellular proteins; lipoprotein; microbes.

Figure 1

SDS-PAGE. Single dimensional gel electrophoresis of extracellular proteins of S. pasteuri isolated from estrus CM. kDa on the right denotes the standard protein molecular weight (MW) marker. kDa on the left shows the approximate MW of high intensity proteins from S. pasteuri. The original figure is provided in the Supplementary File S1 Figure S2 from which lane 5 and the marker lane are used here as the major figure for a better understanding of the exoproteome.

Figure 2

Functional annotation. The identified proteins from S. pasteuri culture supernatant were submitted to the ComparativeGO database, and the proteins were classified according to Biological process (A), Cellular component (B), and Molecular function (C). (A) Proteins were mainly involved in cellular and metabolic activities. (B) The majority of proteins were present in cellular anatomical entities and protein-containing complexes. (C) The major proteins were associated with catalytic and binding activities.

Figure 3

Multiple sequence alignment and phylogenetic tree of Lpp. (A). The alignment of Lpp sequence showed several identical residues predicted with the 4EF1. (B). The evolutionary tree represents clustering pattern of the selected Lpp, MetQ/NlpA and the OBP proteins.

Figure 4

Homology model of Lpp protein. The protein is displayed by molecular 3D view with the representation of a rainbow color pattern from N-terminal to C-terminal (A), and the solid surface representation of Lpp as shown by hydrophobicity surface view (B), and it is best for showing the shape of the pocket. The surface view color pattern indicates most hydrophilic residues in blue, the strongest hydrophobic residues in orange red and the intermediate residues in white.

Figure 5

The validation of the homology modelled Lpp. The model was validated using Ramachandran plot analysis (A), the plot shows the most favoured and allowed residues in the limited phi/psi regions. “*” indicates that only few residues are present in the disallowed region of Ramachandran plot. The Z-score plot (B) shows the Lpp structure similar to native protein.

Figure 6

Binding cavity of Lpp. Hydrophobic residues present in the major and minor cavities of the protein model were visualized. The top 5 binding sites (red, blue, green, purple, orange) were observed using the CASTp server. The first red color-marked cavity shows the larger area and volume in the Lpp protein. Both area and volume shown are solvent-accessible surface area/volume (Lee-Richard molecular surface).

Figure 7

Interactions of the Lpp with the pheromones/volatiles. The 2D residue map of pheromone interactions with the Lpp is shown, and the residual interactions were observed below 4.0 Å. The acetic (CID_176) and butanoic acids (CID_264) have several H-bond interactions (A,B). Propanoic (CID_1032) and isobutyric (CID_6590) acids have hydrophobic and Pi-stacking interactions (C,D). Valeric (CID_7991) and isovaleric (CID_10430) acids exhibit H-bond, hydrophobic, Pi-Sigma and Pi-Alkyl interactions (E,F).