The Role of the Signaling Peptide VP1 in the Virulence of Streptococcus pneumoniae

Abstract

The bacteria Streptococcus pneumoniae, also known as the pneumococcus, is a common and important human pathogen responsible for millions of deaths worldwide.This pathogen can colonize and reside in the back of the nasopharynx asymptomatically as part of the nasopharyngeal resident microflora. From the nasopharynx, the bacteria can disseminate to the middle ear and sinus to cause mild diseases, or spread into the lungs to cause severe diseases like pneumonia. Therefore, Streptococcus pneumoniae represents an important public health concern.In the mucosa, the pneumococcus forms multicellular surface-associated structures called biofilms. These structures play a crucial role in the survival of bacteriaduring infection. Biofilms are recalcitrant to antibiotic treatment, facilitate the adhesion and cohesion of the cells to the host cells and each other, and hide bacterial cells from immune surveillance. Colonization is a population-level behavior, and its success depends on cell to cell communication and host-environment sensing. Colonization is also a prerequisite for dissemination and disease, thus the molecules that control colonization are high-value candidates for therapeutic interventions. In thepneumococcus, there are two well-characterized classes of secreted peptides that mediate signaling. In the first group, a peptide binds to surface-exposed histidine kinasereceptors on the triggering a cascade, while in the second group, the signaling peptide enters the cells and binds directly to a transcription factor. Additionally, signalingpeptides can also be categorized based on sequence features. Some signaling peptides characterized in pneumococci are the competence stimulating peptide (CSP), the bacteriocin inducing peptide (BIP), and the short hydrophobic peptides (SHP) family. The messages carried by these signaling molecules is not fully understood andconstitutes a significant gap in the knowledge of pneumococcal biology. We identified the product of an uncharacterized peptide-encoding gene as aputative signaling molecule using bioinformatics tools. We also found that this gene is also present across multiple pneumococcal strains. We termed the gene productVirulence Peptide 1 (VP1). We determined that VP1 is a signaling peptide, highly induced in the presence of host cells and in vivo, promotes biofilm development, andserves as a potent virulence determinant. To gather insight into the regulation of vp1, we analyzed the genomicorganization of the vp1 neighborhood. We demonstrated that vp1 expression is under the control of a previously uncharacterized transcription factor, the regulator Rgg144.We found that pneumococci also encode a novel peptide belonging to the family of Rggcognate activator peptides SHP, which lay in between rgg144 and vp1 operons. Wefound that the pair Rgg144/SHP144 positively regulates the expression of the vp1 operon. Subsequently, we built on the molecular mechanism of the VP1 function to reveal novel bacterial and host molecules that enhance adherence and colonization. Transcriptional studies revealed that VP1 triggers the expression of operons involved in the transport and metabolism of hyaluronic acid (HA), a glycosaminoglycan present in the host extracellular matrix. HA is a crucial component of the extracellular matrix that ispresent on the apical surface of epithelial cells in the human airways. Previous studies have reported that molecules involved in the acquisition of HA contribute to colonization.Our findings suggest that host HA serves as an anchor for pneumococcal cells and that genes involved in transport and metabolism of HA promote adherence. Finally, ourresults are strongly supported by studies in a murine model of colonization. In conclusion, we describe a molecular pathway utilized by pneumococci to adhere to the host cell and promote colonization. We provide substantial evidence that supports our hypothesis that VP1 is a secreted signaling molecule. Our data indicate that VP1 plays a pivotal role in pneumococcal biofilm development and behavior by orchestrating gene expression. Our studies are implemented using a virulent strain from the clinically relevant PMEN1 lineage. We conclude that VP1 is a novel streptococcal regulatory peptide that controls biofilm development and pneumococcal pathogenesis.Based on our evidence, we propose that VP1 serves as a marker for colonization and a target for drug design.