Targeted control of pneumolysin production by a mobile genetic element in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen that can cause severe invasive diseases such as pneumonia, septicaemia and meningitis. Young children are at a particularly high risk, with an estimated 3-4 million cases of severe disease and between 300 000 and 500 000 deaths attributable to pneumococcal disease each year. The haemolytic toxin pneumolysin (Ply) is a primary virulence factor for this bacterium, yet despite its key role in pathogenesis, immune evasion and transmission, the regulation of Ply production is not well defined. Using a genome-wide association approach, we identified a large number of potential affectors of Ply activity, including a gene acquired horizontally on the antibiotic resistance-conferring Integrative and Conjugative Element (ICE) ICESp23FST81. This gene encodes a novel modular protein, ZomB, which has an N-terminal UvrD-like helicase domain followed by two Cas4-like domains with potent ATP-dependent nuclease activity. We found the regulatory effect of ZomB to be specific for the ply operon, potentially mediated by its high affinity for the BOX repeats encoded therein. Using a murine model of pneumococcal colonization, we further demonstrate that a ZomB mutant strain colonizes both the upper respiratory tract and lungs at higher levels when compared to the wild-type strain. While the antibiotic resistance-conferring aspects of ICESp23FST81 are often credited with contributing to the success of the S. pneumoniae lineages that acquire it, its ability to control the expression of a major virulence factor implicated in bacterial transmission is also likely to have played an important role.

The Breadth and Molecular Basis of Hcp-Driven Type VI Secretion System Effector Delivery.

The type VI secretion system (T6SS) is a bacterial nanoscale weapon that delivers toxins into prey ranging from bacteria and fungi to animal hosts. The cytosolic contractile sheath of the system wraps around stacked hexameric rings of Hcp proteins, which form an inner tube. At the tip of this tube is a puncturing device comprising a trimeric VgrG topped by a monomeric PAAR protein. The number of toxins a single system delivers per firing event remains unknown, since effectors can be loaded on diverse sites of the T6SS apparatus, notably the inner tube and the puncturing device. Each VgrG or PAAR can bind one effector, and additional effector cargoes can be carried in the Hcp ring lumen. While many VgrG- and PAAR-bound toxins have been characterized, to date, very few Hcp-bound effectors are known. Here, we used 3 known Pseudomonas aeruginosa Hcp proteins (Hcp1 to -3), each of which associates with one of the three T6SSs in this organism (H1-T6SS, H2-T6SS, and H3-T6SS), to perform in vivo pulldown assays. We confirmed the known interactions of Hcp1 with Tse1 to -4, further copurified a Hcp1-Tse4 complex, and identified potential novel Hcp1-bound effectors. Moreover, we demonstrated that Hcp2 and Hcp3 can shuttle T6SS cargoes toxic to Escherichia coli. Finally, we used a Tse1-Bla chimera to probe the loading strategy for Hcp passengers and found that while large effectors can be loaded onto Hcp, the formed complex jams the system, abrogating T6SS function. IMPORTANCE The type VI secretion system (T6SS) is an effective weapon used by bacteria to outgrow or kill competitors. It can be used by endogenous commensal microbiota to prevent invasion by pathogens or by pathogens to overcome resident flora and successfully colonize a host or a specific environmental niche. The T6SS is a key contributor to this continuous arms race between organisms as it delivers a multitude of toxins directed at essential processes, such as nucleic acid synthesis and replication, cell wall and membrane integrity, protein synthesis, or cofactor abundance. Many T6SS toxins with unknown function remain to be discovered, whose yet-uncharacterized targets could be exploited for antimicrobial drug design. The systematic search for these toxins is not facilitated by the presence of readily recognizable T6SS motifs, and unbiased screening approaches are thus required. Here, we successfully used a known shuttle for cargo T6SS effectors, Hcp, as bait to identify uncharacterized toxins.