The Autolysin LytA Contributes to Efficient Bacteriophage Progeny Release in Streptococcus pneumoniae

Most bacteriophages (phages) release their progeny through the action of holins that form lesions in the cytoplasmic membrane and lysins that degrade the bacterial peptidoglycan. Although the function of each protein is well established in phages infecting Streptococcus pneumoniae, the role—if any—of the powerful bacterial autolysin LytA in virion release is currently unknown. In this study, deletions of the bacterial and phage lysins were done in lysogenic S. pneumoniae strains, allowing the evaluation of the contribution of each lytic enzyme to phage release through the monitoring of bacterial-culture lysis and phage plaque assays. In addition, we assessed membrane integrity during phage-mediated lysis using flow cytometry to evaluate the regulatory role of holins over the lytic activities. Our data show that LytA is activated at the end of the lytic cycle and that its triggering results from holin-induced membrane permeabilization. In the absence of phage lysin, LytA is able to mediate bacterial lysis and phage release, although exclusive dependence on the autolysin results in reduced virion egress and altered kinetics that may impair phage fitness. Under normal conditions, activation of bacterial LytA, together with the phage lysin, leads to greater phage progeny release. Our findings demonstrate that S. pneumoniae phages use the ubiquitous host autolysin to accomplish an optimal phage exiting strategy.Streptococcus pneumoniae (pneumococcus), a common and important human pathogen, is characterized by the high incidence of lysogeny in isolates associated with infection (34, 44). Pneumococcal bacteriophages (phages) share with the majority of bacteriophages infecting other bacterial species the “holin-lysin” system to lyse the host cell and release their progeny at the end of the lytic cycle. Genes encoding both holins and lysins (historically termed “endolysins”) are indeed found in the genomes of all known pneumococcal phages (8, 28, 31, 37). Supporting this mechanism, a lytic phenotype in the heterologous Escherichia coli system was achieved only by the simultaneous expression of the Ejh holin and the Ejl endolysin of pneumococcal phage EJ-1 (8). When these proteins were independently expressed, cellular lysis was not perceived. Similar results were shown for pneumococcal phage Cp-1, not only in E. coli, but also in the pneumococcus itself (28).Phage lysins destroy the pneumococcal peptidoglycan network due to their muralytic activity, whereas holins have been shown in S. pneumoniae to form nonspecific lesions (8), most likely upon a process of oligomerization in the cytoplasmic membrane, as observed for the E. coli phage λ (13, 14, 43). It was generally proposed that holin lesions allow access of phage lysins to the cell wall (52, 54), as the majority of phage lysins, including the pneumococcal endolysins, lack a typical N-terminal secretory signal sequence and transmembrane domains (8). However, recent evidence also highlights the possibility for a holin-independent targeting of phage lysins to the cell wall, where holin lesions seem to be crucial for the activation of the already externalized phage lysins (42, 50, 51). Regardless of the mechanism operating in S. pneumoniae to activate phage lysins, holin activity compromises membrane integrity.Pneumococcal cells present their own autolytic activity, mainly due to the presence of a powerful bacterial cell wall hydrolase, LytA (an N-acetylmuramoyl-l-alanine-amidase), responsible for bacterial lysis under certain physiological conditions (47). Although other bacterial species also encode peptidoglycan hydrolases, the extensive lysis shortly after entering stationary phase caused by LytA is a unique feature of S. pneumoniae. Interestingly, LytA is translocated across the cytoplasmic membrane to the cell wall—where it remains inactive—in spite of the absence of a canonical N-terminal sequence signal (7). In the cell wall, autolysin activities are tightly regulated by mechanisms that seem to be related to the energized state of the cell membrane. In fact, depolarizing agents are able to trigger autolysis in Bacillus subtilis (16, 17), and bacteriocin-induced depletion of membrane potential triggers autolysis of some species of the genera Lactococcus and Lactobacillus, closely related to streptococci (29). It is therefore possible that the holin-inflicted perturbations of the S. pneumoniae cytoplasmic membrane upon the induction of the lytic cycle may trigger not only the lytic activity of the phage lysin, but also that of inactive LytA located in the cell wall. Accordingly, LytA could also participate in the release of phage particles at the end of the infectious cycle, especially considering its powerful autolytic activity. Previous studies have suggested a role for the host autolytic enzyme in the release of phage progeny (11, 38), but in fact, the evidence is unclear and dubious, considering that the existence of phage-encoded lysins was unknown or very poorly understood and some of the experimental conditions used to show a role of LytA could have also affected the activity of the phage lysin (38).To clarify the possible role of the bacterial autolysin in host lysis, we used the S. pneumoniae strain SVMC28, lysogenic for the SV1 prophage (34), which contains a typical “holin-lysin” cassette, and a different host strain lysogenized with the same SV1 phage. Our results show that LytA is activated by the holin-induced membrane disruption, just like the phage endolysin. In the absence of the endolysin, LytA is capable of mediating host lysis, releasing functional phage particles able to complete their life cycle. Still, sole dependence on LytA results in an altered pattern of phage release that may reduce phage fitness. Importantly, we also show that, together with the endolysin, the concurrent LytA activation is critical for optimal phage progeny release.