Inhibition of Competence Development, Horizontal Gene Transfer and Virulence in Streptococcus pneumoniae by a Modified Competence Stimulating Peptide

Abstract

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae.

Therapeutic potential of theStreptococcus pneumoniaecompetence regulon

“…These findings were confirmed by Oggioni et al, which show that administration of CSP in a mouse model of pneumonia infection increases virulence of pneumococci [13]. By using an opposite strategy, we have most recently shown that certain CSP analogs could competitively inhibit the competence regulon-dependent pneumonia [14]. Collectively, these results suggest that the competence regulon is important for lung infection.…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…More impressively, coinjection of the inhibitor peptide with S. pneumoniae in a mouse lung infection model led to improved mouse survival compared to a no-treatment control or to coinjection of S. pneumoniae with a noninhibitory peptide analogue. Additionally, coinjection of the inhibitor blocked the bacteria's ability to transform into an antibiotic-resistant strain within the mouse when DNA encoding antibiotic resistance was supplied simultaneously (226). These results indicate a potential to block acquisition of new genes by bacteria, provided that an inhibitor is supplied at a time when extracellular DNA is available.…”

“…S. pneumoniae competence is induced by the linear, 17-amino-acid competencestimulating peptide (CSP), which is recognized at the cell surface by the histidine kinase ComD (22). In studies probing critical residues of CSP, alanine substitution of the first amino acid generated a peptide (CSP-E1A) that could block the ability of the native CSP to bind and stimulate ComD signaling (226). During in vitro studies, adding CSP-E1A to cells significantly decreased virulence factor expression.…”

Addiction of Hypertransformable Pneumococcal Isolates to Natural Transformation for In Vivo Fitness and Virulence

“…More importantly, our data, for the first time, have uncovered that natural transformation makes a significantly biased contribution to the in vivo fitness and virulence of highly transformable pneumococcal isolates. Because the previously described importance of the competence system in pneumococcal virulence remains a mystery (8,60), this "addictive" requirement of natural transformation in the hypertransformable pneumococci has provided a new venue to decipher the mechanism(s) by which natural transformation enhances pneumococcal fitness and pathogenesis.…”

“…Natural transformation has also been implicated in allelic replacement or interstrain shuffling of the genes encoding immunoprotective proteins (e.g., PspA, CbpA, and PsrP), leading to antigenic variation and thereby evasion of human immunity (75). Lastly, the competence system has been indicated to be necessary for the carriage and virulence of the pneumococci (8,9,60), although the molecular basis of this requirement is unclear. On the other hand, natural transformation leads to transient growth arrest (78,79), fratricidal killing of noncompetent cells (80), and disruption of beneficial alleles (81).…”