The underlying cause of this difference in bacterial chain length between pneumococci and other streptococci, however, is unclear. among pathogenic microbes. == INTRODUCTION == Streptococcus pneumoniae(the pneumococcus) is a leading cause of invasive bacterial infection (OBrien et al., 2009). This pathogen was previously known asDiplococcus pneumoniaebecause it is typically seen clinically in the form of diplococci or short bacterial chains. This is in contrast to closely related streptococci, however, which typically appear as long bacterial chains. The underlying reason for this difference in bacterial chain length between pneumococci and other streptococci, however, has remained unclear. Pneumococcal infections are controlled by host neutrophils, which kill this pathogen via opsonophagocytosis (OPH), a process that requires opsonization of bacteria by the complement system (Dalia et al., 2010;Lysenko et al., 2007;Matthias et al., 2008). Activation of this system results in the covalent S-Ruxolitinib deposition of complement component 3 (C3) onto bacterial surfaces (Lambris et al., 2008). On gram-negative bacteria, this can lead to direct complement-mediated lysis S-Ruxolitinib of cells, while gram-positives are S-Ruxolitinib resistant to lysis due to their thick peptidoglycan layer. However, C3 can also interact with complement receptors on neutrophils to promote phagocytosis. Deposition of C3 onto pneumococci can result from activation of either the classical or alternative pathways (Brouwer et al., 2008). Activation of the classical pathway can be directed to bacterial surfaces with the aid of antibodies, while the alternative pathway stochastically activates complement on bacterial surfaces. Once opsonized, bacteria can be recognized by surface receptors on neutrophils and ingested by phagocytosis. Once internalized,S. pneumoniaeis efficiently killed in the phagolysosome (Standish and Weiser, 2009). Pneumococci resist opsonization by complement due to their surface capsular polysaccharide (Hyams et al., 2010), which masks underlying structures and activates complement poorly. In Rabbit polyclonal to AKAP5 addition to capsule, the pneumococcus has surface proteins that directly interact with serum components to evade complement and subsequent phagocytosis (Dalia et al., 2010;Jarva et al., 2003). To identify additional factors that promote resistance to this mechanism of killing, we screened a genomic library for mutants that were more sensitive to OPH killing. A common phenotype among mutants identified by this screen was an increase in bacterial chain length (CL). This lead us to hypothesize that minimization of CL inS. pneumoniaeenhances resistance to OPH killing. == RESULTS == == Increased CL enhances susceptibility to OPH killing == A library of pneumococcal mutants was created using the mariner transposon and screened for increased susceptibility to OPH killing by human neutrophils. In addition to genes affecting capsular polysaccharide expression, some of the genes identified by this screen encoded putative cell wall components or regulatory factors (Table S1). A common phenotype among many of these mutants was an increase in bacterial CL (Figure 1, A and B). The size of bacterial chains was defined as their two-dimensional area in phase contrast images and was used as a proxy for CL (Figure 1B). Mutant strains from the screen displayed varying degrees of chain formation, and as CL increased, resistance to OPH killing decreased (Figure 1, B and C) and this trend was highly significant (Figure 1D). A negative control was the BT1 mutant, which contains the mariner transposon, but does not display increased chain length and was not more susceptible to OPH killing (Figure 1, B and C). Since chain formation results from the incomplete cleavage of peptidoglycan between daughter cells following cell division, a positive control for increased CL was a mutant with an in-frame deletion inlytA, which encodes the major cell wall murein hydrolase in the pneumococcus (Figure 1, B and C) (Tomasz, 1968). Growth of pneumococci in varying concentrations of choline, which causes release of LytA and other choline binding proteins from the cell surface, and in sub-inhibitory concentrations of the bacteriostatic antibiotic erythromycin (Erm), also induced chains of increasing length, providing an independent way to confirm the effects of CL (Figure 1, A and C) (Tomasz, 1968). OPH killing assays performed with the WT grown under these conditions or with thelytAmutant (Figure 1, B and C) confirmed that as CL increased, resistance to OPH killing decreased (Figure 1E). == Figure 1. Increased chain length enhances susceptibility to killing by human neutrophilsex vivo. == Chain formation was assessed among mutant strains and growth conditions by phase contrast microscopy (Scale bar = 10 m) (A). Details of bacterial strains and mutants are described inTable S1. Chain formation was induced.