Streptococcus pneumoniae is a frequent member of the microbiota of the human nasopharynx. Colonization of the nasopharyngeal tract is a first and necessary step in the infectious process and often involves the formation of sessile microbial communities by this human pathogen. The ability to grow and persist as biofilms is an advantage for many microorganisms, because biofilm-grown bacteria show reduced susceptibility to antimicrobial agents and hinder recognition by the immune system. The extent of host protection against biofilm-related pneumococcal disease has not been determined yet. Using pneumococcal strains growing as planktonic cultures or as biofilms, we have investigated the recognition of S. pneumoniae by the complement system and its interactions with human neutrophils. Deposition of C3b, the key complement component, was impaired on S. pneumoniae biofilms. In addition, binding of C-reactive protein and the complement component C1q to the pneumococcal surface was reduced in biofilm bacteria, demonstrating that pneumococcal biofilms avoid the activation of the classical complement pathway. In addition, recruitment of factor H, the downregulator of the alternative pathway, was enhanced by S. pneumoniae growing as biofilms. Our results also show that biofilm formation diverts the alternative complement pathway activation by a PspC-mediated mechanism. Furthermore, phagocytosis of pneumococcal biofilms was also impaired. The present study confirms that biofilm formation in S. pneumoniae is an efficient means of evading both the classical and the PspC-dependent alternative complement pathways the host immune system.

In this review we give an update on the mechanisms of naturally acquired immunity against Streptococcus pneumoniae, one of the major human bacterial pathogens that is a common cause of pneumonia, septicaemia, and meningitis. A clear understanding of the natural mechanisms of immunity to S. pneumoniae is necessary to help define why the very young and elderly are at high risk of disease, and for devising new prevention strategies. Recent data has shown that nasopharynx colonization by S. pneumoniae induces antibody responses to protein and capsular antigens in both mice and humans, and also induces Th17 CD4+ cellular immune responses in mice and increases pre-existing responses in humans. These responses are protective, demonstrating that colonization is an immunizing event. We discuss the data from animal models and humans on the relative importance of naturally acquired antibody and Th17 cells on immunity to S. pneumoniae at three different anatomical sites of infection, the nasopharynx (the site of natural asymptomatic carriage), the lung (site of pneumonia), and the blood (site of sepsis). Mouse data suggest that CD4+ Th17 cells prevent both primary and secondary nasopharyngeal carriage with no role for antibody induced by previous colonization. In contrast, antibody is necessary for prevention of sepsis but CD4+ cellular responses are not. Protection against pneumonia requires a combination of both antibody and Th17 cells, in both cases targeting protein rather than capsular antigen. Proof of which immune component prevents human infection is less easily available, but two recent papers demonstrate that human IgG targeting S. pneumoniae protein antigens is highly protective against septicaemia. The role of CD4+ responses to prior nasopharyngeal colonization for protective immunity in humans is unclear. The evidence that there is significant naturally-acquired immunity to S. pneumoniae independent of anti-capsular polysaccharide has clinical implications for the detection of subjects at risk of S. pneumoniae infections, and the data showing the importance of protein antigens as targets for antibody and Th17 mediated immunity should aid the development of new vaccine strategies.

Prophages (viral genomes integrated within a host bacterial genome) can confer various phenotypic traits to their hosts, such as enhanced pathogenicity. Here we analyse >1300 genomes of 70 different Streptococcus species and identify nearly 800 prophages and satellite prophages (prophages that do not encode their own structural components but rely on the bacterial host and another helper prophage for survival). We show that prophages and satellite prophages are widely distributed among streptococci in a structured manner, and constitute two distinct entities with little effective genetic exchange between them. Cross-species transmission of prophages is not uncommon. Furthermore, a satellite prophage is associated with virulence in a mouse model of Streptococcus pneumoniae infection. Our findings highlight the potential importance of prophages in streptococcal biology and pathogenesis.

Streptococcus pneumoniae is a common colonizer of the human nasopharynx and one of the major pathogens causing invasive disease worldwide. Dissection of the molecular pathways responsible for colonization, invasion, and evasion of the immune system will provide new targets for antimicrobial or vaccine therapies for this common pathogen. We have constructed mutants lacking the pneumococcal cell wall hydrolases (CWHs) LytB and LytC to investigate the role of these proteins in different phases of the pneumococcal pathogenesis. Our results show that LytB and LytC are involved in the attachment of S. pneumoniae to human nasopharyngeal cells both in vitro and in vivo. The interaction of both proteins with phagocytic cells demonstrated that LytB and LytC act in concert avoiding pneumococcal phagocytosis mediated by neutrophils and alveolar macrophages. Furthermore, C3b deposition was increased on the lytC mutant confirming that LytC is involved in complement evasion. As a result, the lytC mutant showed a reduced ability to successfully cause pneumococcal pneumonia and sepsis. Bacterial mutants lacking both LytB and LytC showed a dramatically impaired attachment to nasopharyngeal cells as well as a marked degree of attenuation in a mouse model of colonization. In addition, C3b deposition and phagocytosis was more efficient for the double lytB lytC mutant and its virulence was greatly impaired in both systemic and pulmonary models of infection. This study confirms that the CWHs LytB and LytC of S. pneumoniae are essential virulence factors involved in the colonization of the nasopharynx and in the progress of invasive disease by avoiding host immunity.

The complement system is a key component of the host immune response for the recognition and clearance of Streptococcus pneumoniae . In this study, we demonstrate that the amidase LytA, the main pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein to S. pneumoniae , thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for the lytA ply double mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia.

Secondary bacterial infections are an important cause of morbidity and mortality associated with influenza infections. As bacterial disease can be caused by a disturbance of the host microbiome, we examined the impact of influenza on the upper respiratory tract microbiome in a human challenge study. The dynamics and ecology of the throat microbiome were examined following an experimental influenza challenge of 52 previously-healthy adult volunteers with influenza A/Wisconsin/67/2005 (H3N2) by intranasal inoculation; 35 healthy control subjects were not subjected to the viral challenge. Serial oropharyngeal samples were taken over a 30-day period, and the V1-V3 region of the bacterial 16S ribosomal RNA sequences were amplified and sequenced to determine the composition of the microbiome. The carriage of pathogens was also detected. Of the 52 challenged individuals, 43 developed proven influenza infections, 33 of whom became symptomatic. None of the controls developed influenza, although 22% reported symptoms. The diversity of bacterial communities remained remarkably stable following the acquisition of influenza, with no significant differences over time between individuals with influenza and those in the control group. Influenza infection was not associated with perturbation of the microbiome at the level of phylum or genus. There was no change in colonization rates with Streptococcus pneumoniae or Neisseria meningitidis. The throat microbiota is resilient to influenza infection, indicating the robustness of the upper-airway microbiome.