Identification and Functional Characterization of Peptides With Antimicrobial Activity From the Syphilis Spirochete, Treponema pallidum

Abstract

The etiological agent of syphilis, Treponema pallidum ssp. pallidum, is a highly invasive “stealth” pathogen that can evade the host immune response and persist within the host for decades. This obligate human pathogen is adept at establishing infection and surviving at sites within the host that have a multitude of competing microbes, sometimes including pathogens. One survival strategy employed by bacteria found at polymicrobial sites is elimination of competing microorganisms by production of antimicrobial peptides (AMPs). Antimicrobial peptides are low molecular weight proteins (miniproteins) that function directly via inhibition and killing of microbes and/or indirectly via modulation of the host immune response, which can facilitate immune evasion. In the current study, we used bioinformatics to show that approximately 7% of the T. pallidum proteome is comprised of miniproteins of 150 amino acids or less with unknown functions. To investigate the possibility that AMP production is an unrecognized defense strategy used by T. pallidum during infection, we developed a bioinformatics pipeline to analyze the complement of T. pallidum miniproteins of unknown function for the identification of potential AMPs. This analysis identified 45 T. pallidum AMP candidates; of these, Tp0451a and Tp0749 were subjected to further bioinformatic analyses to identify AMP critical core regions (AMPCCRs). Four potential AMPCCRs from the two predicted AMPs were identified and peptides corresponding to these AMPCCRs were experimentally confirmed to exhibit bacteriostatic and bactericidal activity against a panel of biologically relevant Gram-positive and Gram-negative bacteria. Immunomodulation assays performed under inflammatory conditions demonstrated that one of the AMPCCRs was also capable of differentially regulating expression of two pro-inflammatory chemokines [monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8)]. These findings demonstrate proof-of-concept for our developed AMP identification pipeline and are consistent with the novel concept that T. pallidum expresses AMPs to defend against competing microbes and modulate the host immune response.

In-Depth Proteome Coverage of In Vitro-Cultured Treponema pallidum and Quantitative Comparison Analyses with In Vivo-Grown Treponemes

“…In addition, 68% of these undetected proteins were either classified as “function unknown” or could not be assigned a function using COG , analysis (Figure and Table S5). In accordance with our previous findings from in vivo-grown treponemes, a high proportion (60%) of the undetected proteins were also found to be miniproteins comprising less than 150 amino acids that often contain high numbers of lysine and arginine residues . This results in the production of particularly small trypsin-cleaved peptides during proteomics sample preparation that are below the size detection limit of most mass spectrometers.…”

“…In the current study, we were able to detect 39 of the 68 miniproteins of unknown function, which, when combined with the previous proteomics studies, − enabled the identification of 62% of this protein subgroup (Figure C,D, Tables and S12). Although we were able to detect more miniproteins in the present study compared to the in vivo study, only the same three miniproteins were detected as high-abundance proteins in both studies: TPANIC_0084, TPANIC_0777, and the predicted AMP, TPANIC_0847 …”

“…However, only a limited number of proteins from this category were identified as high-abundance proteins from either growth condition (Table S9 and ref ). Sixty-eight miniproteins, two of which have been shown to exhibit antimicrobial peptide (AMP) activities, comprise a subgroup of the treponemal proteins of unknown function, which comprise 150 amino acids or less. In the current study, we were able to detect 39 of the 68 miniproteins of unknown function, which, when combined with the previous proteomics studies, − enabled the identification of 62% of this protein subgroup (Figure C,D, Tables and S12).…”

“…Although we were able to detect more miniproteins in the present study compared to the in vivo study, 25 only the same three miniproteins were detected as high-abundance proteins in both studies: TPANIC_0084, TPANIC_0777, and the predicted AMP, TPANIC_0847. 49 In our preceding T. pallidum global proteomics study, we compiled a list of 34 proteins, identified from the literature, 25 which comprised experimentally confirmed and predicted T. pallidum surface-exposed OMPs. 16,26,27,51,52,59−72 In the present study, we were able to detect the protein expression of 31 known and predicted treponemal OMPs, which increased the combined OMP repertoire coverage to 94% (Figure 8A,B, and Tables 1 and 2).…”

“…In accordance with our previous findings from in vivo-grown treponemes, 25 a high proportion (60%) of the undetected proteins were also found to be miniproteins comprising less than 150 amino acids that often contain high numbers of lysine and arginine residues. 49 This results in the production of particularly small trypsin-cleaved peptides during proteomics sample preparation that are below the size detection limit of most mass spectrometers. Therefore, these physicochemical properties are likely important contributing factors that limit the detection of this protein class by MS. 25 To determine the tendency for a subcellular compartment bias, the cellular locations of the 62 undetected T. pallidum proteins were predicted using the bacterial localization prediction tool PSORTb 3.0.3 (https://www.psort.org/ psortb/) 50 and the subcellular location annotations from UniProt Archive (UniParc) searches (https://www.uniprot.…”