Identification of Pneumococcal Factors Affecting Pneumococcal Shedding Shows that the dlt Locus Promotes Inflammation and Transmission

Abstract

Host-to-host transmission is a necessary but poorly understood aspect of microbial pathogenesis. Herein, we screened a genomic library of mutants of the leading respiratory pathogen Streptococcus pneumoniae generated by mariner transposon mutagenesis (Tn-Seq) to identify genes contributing to its exit or shedding from the upper respiratory tract (URT), the limiting step in the organism’s transmission in an infant mouse model. Our analysis focused on genes affecting the bacterial surface that directly impact interactions with the host. Among the multiple factors identified was the dlt locus, which adds d-alanine onto lipoteichoic acids (LTA) and thereby increases Toll-like receptor 2-mediated inflammation and resistance to antimicrobial peptides. The more robust proinflammatory response in the presence of d-alanylation promotes secretions that facilitate pneumococcal shedding and allows for transmission. Expression of the dlt locus is controlled by the CiaRH system, which senses cell wall stress in response to antimicrobial activity, including in response to lysozyme, the most abundant antimicrobial along the URT mucosa. Accordingly, in a lysM−/− host, there was no longer an effect of the dlt locus on pneumococcal shedding. Thus, our findings demonstrate how a pathogen senses the URT milieu and then modifies its surface characteristics to take advantage of the host response for transit to another host. IMPORTANCE Streptococcus pneumoniae (the pneumococcus) is a common cause of respiratory tract and invasive infection. The overall effectiveness of immunization with the organism’s capsular polysaccharide depends on its ability to block colonization of the upper respiratory tract and thereby prevent host-to-host transmission. Because of the limited coverage of current pneumococcal vaccines, we carried out an unbiased in vivo transposon mutagenesis screen to identify pneumococcal factors other than its capsular polysaccharide that affect transmission. One such candidate was expressed by the dlt locus, previously shown to add d-alanine onto the pneumococcal lipoteichoic acid present on the bacterial cell surface. This modification protects against host antimicrobials and augments host inflammatory responses. The latter increases secretions and bacterial shedding from the upper respiratory tract to allow for transmission. Thus, this study provides insight into a mechanism employed by the pneumococcus to successfully transit from one host to another.

Experimental Evolutionin vivoto Identify Selective Pressures During Pneumococcal Colonization

“…We hypothesize this gene is maintained because its deletion is associated with increased sensitivity to antimicrobial peptides and thus reduces fitness in niches with significant inflammatory infiltrate, such as the pneumonic lung. In addition, recent studies have also demonstrated a role of dltB in pneumococcal transmission whereby mutants in this locus have significantly reduced shedding from colonized mice which reduces mammalian transmission 10 . The increased adhesion and colonization phenotypes conferred by loss of this locus may initially be beneficial, but the failure to transmit to new hosts may explain the retention of this gene in S. pneumoniae given the fitness advantages we observe during colonization.…”

“…This is one of the major selective pressures on the pneumococcus, as invasive disease is typically envisioned as an evolutionary dead-end due to clearance by the immune system or antibiotics or via the death of the host, thereby preventing further transmission of the strain. As such, the pneumococcus has evolved numerous strategies to facilitate efficient transmission and colonization of new hosts [8][9][10][11][12][13] . Mutational screens have identified many pneumococcal genes that are required for successful colonization of the nasal passages as well as for invasive infection of the lungs and the bloodstream 14,15 .…”

“…Using whole-population, whole-genomic sequencing and new methods to infer genotype structure, we were able to track individual mutations and the rise of different haplotypes in the pneumococcal population during repeated passages in murine hosts. We identified several parallel, null mutations in dltB, responsible for incorporation of D-alanine into teichoic acids on the bacterial surface that was under strong positive selective pressure 10 . Subsequent deletion of dltB resulted in a fitness benefit during experimental murine nasal colonization and enhanced bacterial adherence to cultured epithelial cells.…”

Competitive exclusion strengthens selection for transmissibility and increases the benefit of recombination for within-host adaptation

“…The balance between transmissibility and within-host fitness parallels an invasion-persistence trade-off in other organisms—some genotypes may more readily disperse and colonize new hosts/territories, but are less persistent and thus fare poorly when present in the same environment as less dispersable but more competitive genotypes. This trade-off is evident in several S. pneumoniae genes, such as toxin pneumolysin [40, 41], capsule serotype (for example, serotype 4 compared to 23F) [40, 42], and the aforementioned dltB [30, 43], which promote transmissibility at the expense of within-host fitness. Whether these genes are favored by selection and maintained depends on the relative importance of transmissibility and within-host fitness, as shown by the loss of dltB in serial passage where transmission is more certain.…”

Resistance Mechanisms to Antimicrobial Peptides in Gram-Positive Bacteria

“…Cell wall modifications in pneumococci can be triggered by AMPs. Treatment with lysozyme leads to upregulation of the dlt locus through the CiaRH sensoring system, resulting in lipoteichoic acid (LTA) modifications and increased inflammatory responses, which in that case contributed to bacterial shedding and transmission (Zafar et al, 2019). Thus, D-alanylation of the cell wall -a mechanism of AMP resistance shared among different Gram-positive microbes-can be induced, in pneumococci, by treatment with antimicrobial proteins that target the bacterial cell wall.…”