Aras Kadioglu

The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease.

Streptococcus pneumoniae is a Gram-positive bacterial pathogen that colonizes the mucosal surfaces of the host nasopharynx and upper airway. Through a combination of virulence-factor activity and an ability to evade the early components of the host immune response, this organism can spread from the upper respiratory tract to the sterile regions of the lower respiratory tract, which leads to pneumonia. In this Review, we describe how S. pneumoniae uses its armamentarium of virulence factors to colonize the upper and lower respiratory tracts of the host and cause disease.

Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.

Switch from planktonic to sessile life : a major event in pneumococcal pathogenesis

Two main patterns of gene expression of Streptococcus pneumoniae were observed during infection in the host by quantitative real time RT‐PCR; one was characteristic of bacteria in blood and one of bacteria in tissue, such as brain and lung. Gene expression in blood was characterized by increased expression of pneumolysin, pspA and hrcA, while pneumococci in tissue infection showed increased expression of neuraminidases, metalloproteinases, oxidative stress and competence genes. In vitro situations with similar expression patterns were detected in liquid culture and in a newly developed pneumococcal model of biofilm respectively. The biofilm model was dependent on addition of synthetic competence stimulating peptide (CSP) and no biofilm was formed by CSP receptor mutants. As one of the differentially expressed gene sets in vivo were the competence genes, we exploited competence‐specific tools to intervene on pneumococcal virulence during infection. Induction of the competence system by the quorum‐sensing peptide, CSP, not only induced biofilm formation in vitro, but also increased virulence in pneumonia in vivo. In contrast, a mutant for the ComD receptor, which did not form biofilm, also showed reduced virulence in pneumonia. These results were opposite to those found in a bacteraemic sepsis model of infection, where the competence system was downregulated. When pneumococci in the different physiological states were used directly for challenge, sessile cells grown in a biofilm were more effective in inducing meningitis and pneumonia, while planktonic cells from liquid culture were more effective in inducing sepsis. Our data enable us, using in vivo gene expression and in vivo modulation of virulence, to postulate the distinction – from the pneumococcal point of view – between two main types of disease. During bacteraemic sepsis pneumococci resemble planktonic growth, while during tissue infection, such as pneumonia or meningitis, pneumococci are in a biofilm‐like state.

Host cellular immune response to pneumococcal lung infection in mice.

ABSTRACT Although there is substantial evidence that pneumolysin is an important virulence factor in pneumococcal pneumonia, relatively little is known about how it influences cellular infiltration into the lungs. We investigated how the inability of mutant pneumococci to produce pneumolysin altered the pattern of inflammation and cellular infiltration into the lungs. The effect on bacterial growth in the lungs also was assessed. There were three phases of growth of wild-type bacteria in the lungs: a decline followed by a rapid increase and then stasis or decline. The absence of pneumolysin was associated with a more rapid early decline and then a much slower increase in numbers. The pattern of inflammatory-cell accumulation also had distinct stages, and the timing of these stages was influenced by the presence of pneumolysin. Neutrophils began to accumulate about 12 to 16 h after infection with wild-type pneumococci. This accumulation occurred after the early decline in pneumococcal numbers but coincided with the period of rapid growth. Following infection with pneumococci unable to make pneumolysin, neutrophil influx was slower and less intense. Coincident with the third stage of pneumococcal growth was an accumulation of T and B lymphocytes at the sites of inflammation, but the accumulation was not associated with an increase in the total number of lymphocytes in the lungs. Lymphocyte accumulation in the absence of pneumolysin occurred but was delayed.

Alternatives to antibiotics—a pipeline portfolio review

Antibiotics have saved countless lives and enabled the development of modern medicine over the past 70 years. However, it is clear that the success of antibiotics might only have been temporary and we now expect a long-term and perhaps never-ending challenge to find new therapies to combat antibiotic-resistant bacteria. A broader approach to address bacterial infection is needed. In this Review, we discuss alternatives to antibiotics, which we defined as non-compound approaches (products other than classic antibacterial agents) that target bacteria or any approaches that target the host. The most advanced approaches are antibodies, probiotics, and vaccines in phase 2 and phase 3 trials. This first wave of alternatives to antibiotics will probably best serve as adjunctive or preventive therapies, which suggests that conventional antibiotics are still needed. Funding of more than £1·5 billion is needed over 10 years to test and develop these alternatives to antibiotics. Investment needs to be partnered with translational expertise and targeted to support the validation of these approaches in phase 2 trials, which would be a catalyst for active engagement and investment by the pharmaceutical and biotechnology industry. Only a sustained, concerted, and coordinated international effort will provide the solutions needed for the future.

The role of pneumolysin in pneumococcal pneumonia and meningitis

Diseases caused by Streptococcus pneumoniae include pneumonia, septicaemia and meningitis. All these are associated with high morbidity and mortality. The pneumococcus can colonize the nasopharynx, and this can be a prelude to bronchopneumonia and invasion of the vasculature space. Proliferation in the blood can result in a breach of the blood–brain barrier and entry into the cerebrospinal fluid (CSF) where the bacteria cause inflammation of the meningeal membranes resulting in meningitis. The infected host may develop septicaemia and/or meningitis secondary to bronchopneumonia. Also septicaemia is a common precursor of meningitis. The mechanisms surrounding the sequence of infection are unknown, but will be dependent on the properties of both the host and bacterium. Treatment of these diseases with antibiotics leads to clearance of the bacteria from the infected tissues, but the bacteriolytic nature of antibiotics leads to an acute release of bacterial toxins and thus after antibiotic therapy the patients can be left with organ‐specific deficits. One of the main toxins released from pneumococci is the membrane pore forming toxin pneumolysin. Here we review the extensive studies on the role of pneumolysin in the pathogenesis of pneumococcal diseases.

Pneumococcal Neuraminidases A and B Both Have Essential Roles during Infection of the Respiratory Tract and Sepsis

ABSTRACT We examined the role of the neuraminidases NanA and NanB in colonization and infection in the upper and lower respiratory tract by Streptococcus pneumoniae, as well as the role of these neuraminidases in the onset and development of septicemia following both intranasal and intravenous infection. We demonstrated for the first time using outbred MF1 mouse models of infection that both NanA and NanB were essential for the successful colonization and infection of the upper and lower respiratory tract, respectively, as well as pneumococcal survival in nonmucosal sites, such as the blood. Our studies have shown that in vivo a neuraminidase A mutant is cleared from the nasopharynx, trachea, and lungs within 12 h postinfection, while a neuraminidase B mutant persists but does not increase in either the nasopharynx, trachea, or lungs. We also demonstrated both neuraminidase mutants were unable to cause sepsis following intranasal infections. When administered intravenously, however, both mutants survived initially but were unable to persist in the blood beyond 48 h postinfection and were progressively cleared. The work presented here demonstrates the importance of pneumococcal neuraminidase A and for the first time neuraminidase B in the development of upper and lower respiratory tract infection and sepsis.

CD4-T-Lymphocyte Interactions with Pneumolysin and Pneumococci Suggest a Crucial Protective Role in the Host Response to Pneumococcal Infection

ABSTRACT Previously, we had shown that T cells accumulated in peribronchiolar and perivascular areas of lungs soon after intranasal infection with Streptococcus pneumoniae. We have now presented new evidence, using major histocompatibility class II-deficient mice, that CD4 cells are important for early protective immunity. In addition, we have also shown that a population of human CD4 cells migrates towards pneumococci and that in vivo-passaged pneumococci are substantially more potent at inducing migration than in vitro-grown bacteria. This migratory process is unique to a specific population of CD4 cells, is highly reproducible, and is independent of prior CD4 cell activation, and yet the migratory process results in a significant proportion of CD4 cells becoming activated. The production of pneumolysin is a key facet in the induction of migration of CD4 cells by in vivo bacteria, as pneumolysin-deficient bacteria do not induce migration, but the data also show that pneumolysin alone is not sufficient to explain the enhanced migration. Increased CD25 expression occurs during migration, and a higher percentage of cells in the migrated population express gamma interferon or interleukin 4 (IL-4) than in the population that did not migrate. There is evidence that the activation of IL-4 expression occurs during migration.

Role of Manganese-Containing Superoxide Dismutase in Oxidative Stress and Virulence of Streptococcus pneumoniae

ABSTRACT Streptococcus pneumoniae was shown to contain two types of superoxide dismutase, MnSOD and FeSOD. Levels of MnSOD increased during growth in an aerobic environment. The sodA gene, encoding MnSOD, of virulent S. pneumoniae type 2 strain D39 was inactivated to give mutant D39HY1. Aerobically, D39HY1 had a lower growth rate than the wild type and exhibited susceptibility to the redox-active compound paraquat, but anaerobic growth of D39HY1 was identical to that of the wild type. Virulence studies showed that the median survival time of mice infected intranasally with D39HY1 was significantly longer than that of mice infected with the wild-type pneumococcus. In contrast to the wild type, D39HY1 did not multiply in lungs during the first 24 h but thereafter grew at the same rate as the wild type. Appearance in the bloodstream was also delayed, but growth in the blood was unimpaired by the sodA mutation. The pattern of inflammation in lungs infected with D39HY1 differed from that in wild-type-infected mice. After infection with D39HY1, neutrophils were densely packed around bronchioles, in contrast to the wild-type infection, where neutrophils were more diffusely localized.

Sialic acid: a preventable signal for pneumococcal biofilm formation, colonization, and invasion of the host.

The correlation between carbohydrate availability, pneumococcal biofilm formation, nasopharyngeal colonization, and invasion of the host has been investigated. Of a series of sugars, only sialic acid (i.e., N-acetylneuraminic acid) enhanced pneumococcal biofilm formation in vitro, at concentrations similar to those of free sialic acid in human saliva. In a murine model of pneumococcal carriage, intranasal inoculation of sialic acid significantly increased pneumococcal counts in the nasopharynx and instigated translocation of pneumococci to the lungs. Competition of both sialic acid-dependent phenotypes was found to be successful when evaluated using the neuraminidase inhibitors DANA (i.e., 2,3-didehydro-2-deoxy-N-acetylneuraminic acid), zanamivir, and oseltamivir. The association between levels of free sialic acid on mucosae, pneumococcal colonization, and development of invasive disease shows how a host-derived molecule can influence a colonizing microbe and also highlights a molecular mechanism that explains the epidemiologic correlation between respiratory infections due to neuraminidase-bearing viruses and bacterial pneumonia. The data provide a new paradigm for the role of a host compound in infectious diseases and point to new treatment strategies.