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Enfermedad neumocónica invasiva: mecanismos moleculares de patogenicidad y protección

AuthorsRamos-Sevillano, Elisa
AdvisorGarcía, Ernesto ; Yuste, José
KeywordsStreptococcus pneumoniae
Virulence factors
Choline-binding proteins
Invasive disease
Issue Date2013
AbstractINTRODUCTION: Streptococcus pneumoniae, the pneumococcus, is a major cause of bacterial sepsis and the most common etiologic agent of acute otitis media, community-acquired pneumonia as well as non-epidemic bacterial meningitis (Bogaert et al., 2004; Wardlaw et al., 2006). Pneumococcal disease is preceded by colonization, which is particularly common in children, with more than one serotype frequently colonizing the nasopharynx of the same individual at the same time (Bogaert et al., 2004). Direct bacterial translocation from the nasopharynx to the bloodstream, generally known as occult bacteraemia, is a well-recognized complication of pneumococcal carriage, particularly in early childhood (Weiser, 2010). More than 25% of the 57 million annual deaths worldwide are estimated to be directly related to infectious diseases. Particularly, respiratory infections are responsible of the death of 4 million people every year. According to World Health Organization (WHO) estimates of sepsis or pneumonia in neonates and pneumonia in older children accounted for 29% of the 10.6 million yearly deaths in children younger than 5 years (10% and 19%, respectively), being Streptococcus pneumoniae the most common cause of severe pneumonia among children in developing countries (http://www.unicef.org/ spanish/publications/file /Pneumonia_The_Forgotten_Killer_of_Children.pdf) as well as in children and adults in Europe and the United States. Overall, the mortality rate of pneumococcal infections is higher than that originated by any other pathogenic bacteria. As a consequence, WHO and organizations such as the Wellcome Trust and the Bill & Melinda Gates Foundation consider the pneumococcal disease as a global burden. Disappointingly, there is much about the pathogenesis of S. pneumoniae meningitis and other invasive pneumococcal diseases which we still do not understand. This thesis prioritizes research on invasive pneumococcal disease at two levels: molecular basis of diseases and development of new therapies. One of the main goals of the proposed research is to carry out a detailed study of some of the host-pathogen interactions that take place during the establishment and development of pneumococcal disease. For this purpose, the roles of different cell wall hydrolases (CWHs) in different phases of the pneumococcal disease (nasopharyngeal colonization, pneumonia and sepsis) have been studied using cell cultures and murine infection models. Several virulence factors of S. pneumoniae as well as cellular receptors and host defence mechanisms of the immune response have been analysed in detail.
Although the capsular polysaccharide (CPS) of S. pneumoniae is a sine qua non requisite for virulence (López y García, 2004), there are increasing evidences showing that several pneumococcal surface proteins play important roles in pathogenesis although with largely unknown functions. Among them, one of the main goals of this Thesis was to investigate CWHs, i.e., LytA, LytB,and LytC, that belong to the family of the so-called choline-binding proteins. These proteins are attached to the bacterial surface through non-covalent interactions with the phosphorylcholine residues present in teichoic and lipoteichoic acids. CWHs are surface proteins that cleave specific covalent bonds of the cell wall, and eventually, cause the lysis and death of the bacteria (López y García, 2004). Among these proteins, LytB is a CWH located closely to the polar ends of the cell. This enzyme has N-acetylglucosaminidase activity and plays an essential role in daughter cell separation (García et al., 1999b; De las Rivas et al., 2002). Moreover, it has been suggested that LytB, regardless of being polymorphic (Moscoso et al., 2005), might be a promising target for the development of a universal pneumococcal vaccine because LytB antibodies significantly protected mice from a lethal challenge with different pneumococcal strains (Wizemann et al., 2001). Other well-characterized CWHs include the LytC lysozyme that, in contrast to LytB, has a general cell-surface distribution (Pérez-Dorado et al., 2010). LytC has autolytic activity at 30°C and the fact that it has its maximum enzymatic activity at this temperature suggests that it might be more crucial in the upper respiratory tract (García et al., 1999a). LytC is one of the major bacterial components that enable S. pneumoniae to lyse non-competent pneumococci (fratricide) (Eldholm et al., 2009). Indeed, fratricide has been proposed as a mechanism of predation that contributes to virulence by regulating the release of several virulence factors.Although much work has been done under in vitro conditions, whether CWHs are important virulence factors involved in direct interaction with the host surfaces and/or in the establishment of pneumococcal disease has not been clearly defined. As a common colonizer of the upper respiratory tract, S. pneumoniae has developed an arsenal of components that are of great importance for biofilm formation and efficient colonization of the nasopharynx, which is the first step of pneumococcal virulence (Domenech et al., 2012). Inactivation of LytB and LytC has been shown to hinder biofilm formation whereas simultaneous disruption of both CWHs markedly reduced biofilm establishment suggesting that both enzymes are important pneumococcal components with additive (or synergistic) effects upon bacterial adhesion (Moscoso et al., 2006; Domenech et al., 2013).
Despite appropriate antibiotic treatment, invasive pneumococcal disease (IPD) is associated to high rates of morbidity and mortality worldwide. A major threat to fight IPD is the appearance of strains resistant to high levels of different antibiotic. Immunization is a safe and highly efficient approach to preventing IPD. Activation of complement cascades by specific antibodies, leads to the formation of the key component C3b that is crucial in host defence against pneumococcus by coating the microorganism and stimulating phagocytosis. In cases where the invading pathogen displays multidrug resistance, antimicrobial concentrations in serum may be insufficient and, therefore, the outcome of the infection will largely depend on the interaction between bacterial virulence factors and host immune mechanisms. One of the major aims of the current study was to investigate the activation of complement immunity and phagocytosis against three multiresistant, clinical pneumococcal isolates in the presence of specific antibodies and subinhibitory concentrations of different antibiotics. PRINCIPAL FINDINGS: In this study, we have constructed isogenic mutants lacking LytB and/or LytC to analyse their role in the engagement and persistence of S. pneumoniae in the upper respiratory tract. We have also investigated the role of these proteins in evasion of complement immunity and phagocytosis. Moreover, we have explored the implication of LytB and LytC in the establishment of pneumococcal pneumonia and bacterial dissemination throughout the systemic circulation. Our results showed 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 or 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 were more efficient for the double lytB lytC mutant and its virulence was greatly impaired in both systemic and pulmonary models of infection. This study confirmed 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.
Among other pneumococcal proteins that are involved in different steps of the pathogenesis process (Kadioglu et al., 2008), pneumolysin (Ply) is a member of the cholesterol-dependent cytolysin family and plays a significant role in virulence by attacking cholesterol-containing membranes (Marriott et al., 2008). In addition, Ply interacts with different components of the host immune response such as TLR4 and complement-mediated immunity modifying the inflammatory response and the recognition of S. pneumoniae by the first complement component of the classical pathway (C1q) (Yuste et al., 2005). The release of Ply from the cytoplasm is mainly mediated by the main autolytic enzyme of the bacterium, LytA, which cleaves the N‑acetylmuramyl‑L-alanine bond of pneumococcal peptidoglycan. LytA participates in several physiologically important phenomena of S. pneumoniae, e.g., cell separation, autolysis during the stationary phase of growth, lysis upon exposure to penicillin and other cell wall inhibitors, virulence, liberation of progeny bacteriophages, etc. (López y García, 2004). Several studies have shown that lytA mutants have attenuated virulence in animal models compared with isogenic wild type strains, but some results are controversial. LytA appears to affect pneumonia but no sepsis, although the mechanism is not well defined yet. The contribution of LytA to virulence is thought to be mediated by its function in the release of Ply and inflammatory mediators such as teichoic acids and peptidoglycan fragments from lysed bacterial cells (Canvin et al., 1995). There exist different virulence factors including the polysaccharide capsule and several proteins of S. pneumoniae that are involved in the evasion of complement-mediated immunity. This microorganism uses different strategies including impaired activation and increased inactivation by recruiting complement fluid-phase down-regulators to diminish the protective role of the complement system (Kadioglu et al., 2008; van der Poll y Opal, 2009). Leukocytes play an important role in inflammatory and immune responses. Bacterial clearance depends on the efficacy of different receptors on the surface of phagocytic cells to recognize, internalize and kill the pathogen (Arredouani et al., 2004; Lanoue et al., 2004; Ku et al., 2007). The initial phases of leukocytes migration to the site of infection after an inflammatory event caused by a microbial pathogen, involve leukocyte rolling that is mediated by interactions of selectins and selectin-ligand molecules (Vicente-Manzanares y Sánchez-Madrid, 2004). Expression of P- and E-selectins by the endothelium is involved in the rolling process and also supports protection against invading pathogens such as S. pneumoniae (Munoz et al., 1997). P-selectin glycoprotein ligand-1 (PSGL-1) mediates leukocyte interactions with P- and E-selectins expressed by the endothelium. It has been shown that PSGL-1 is a receptor for adhesion and entry in neutrophils used by several intracellular pathogens (Rikihisa, 2011) although its phagocytic role in S. pneumoniae infections is unknown.
The main aim of this part of the Thesis was to investigate the role of Ply and LytA in essential aspects of the pathogenesis process including host immune response evasion and pneumococcal dissemination. We show here that LytA avoids complement-mediated immunity by a complex mechanism of impaired activation, increased down-regulation and direct complement degradation. C3b deposition and phagocytosis of S. pneumoniae was greatly enhanced in the absence of LytA and pneumolysin demonstrating a synergistic effect by both proteins in host immune response evasion. Finally, a novel interaction with PSGL-1 was observed, with a significant involvement of LytA in this process. Bacterial levels were significantly increased in PSGL-1–/– knockout mice confirming the importance of this receptor in the recognition and clearance of S. pneumoniae. The successful outcome of infections caused by S. pneumoniae in humans depends on the humoral arm of the immune system (since opsonin/antibody dependent phagocytosis is the major defence mechanism against S. pneumoniae) as well as on an adequate antibiotic treatment. Natural defences and antibiotics may act concomitantly when using drugs as -lactams acting on the cell wall that anchors the capsule. Since colonization is, to some extent, a B-cell immunizing event (Weinverger et al., 2008) and preventive measures as pneumococcal vaccination are increasingly being used, antibodies to capsular polysaccharides (a surrogate marker of immunity) are likely to appear before infection. All these facts were explored in previous murine sepsis models (using a penicillin-resistant 6B S. pneumoniae isolate as infecting agent) where bacteraemia with high colony counts was related with mortality, and both (bacteraemia and mortality) decreased in a dose-related trend by administration of hyperimmune serum or -lactams (Yuste et al., 2002a, b). When combined therapies (hyperimmune serum plus -lactam) were explored, the strategy based on passive immunization (prior to infection) followed by post-infection administration of -lactam was more effective than non-immune serum plus post-infection administered antibiotic therapies (Yuste et al., 2002b). β-Lactams have little effect on the chemotaxis of neutrophils towards areas of infection. However, some of them can increase the oxidative burst of neutrophils by scavenging oxidative species or by inhibition of myeloperoxidase (Labro, 2000). Enhancing this oxidative burst response by -lactams may render some bacterial species more susceptible to phagocytic killing, although the mechanism is not fully understood. In this sense, we have previously reported that the combined effect of amoxicillin or cefotaxime with specific antibodies increases the rate of bacterial clearance in a mouse model (Yuste et al., 2001). In addition, the presence of anti-pneumococcal antibodies led to therapeutic efficacy with subinhibitory concentrations of -lactam antibiotics (Casal et al., 2002).
Moreover, phagocytosis mediated by human or mouse neutrophils was increased when antibiotic-resistant pneumococcal strains were incubated with serum containing specific antibodies and sub-MIC concentrations of -lactams (Cafini et al., 2010). Whether the synergism between anti-pneumococcal antibodies and -lactams is due to a direct interaction of the antimicrobial drug with the host immune system is unclear. To determine whether this effect is partially due to increased recognition of S. pneumoniae by the complement system and whether it might be extended to other groups of antibiotics was another goal of this Thesis. We used three pneumococcal isolates (strains 1515, 69 and 48) of different serotypes and levels of susceptibility/resistance. Hyperimmune serum (HS) was obtained from mice immunized with heat-inactivated strains. Phagocytosis by HS in the presence (or in the absence) of subinhibitory concentrations of antibiotics was measured. To determine the minimal protective and the non-protective (np) highest dilutions/doses over 7 days, in vivo dose-ranging experiments with HS and cefditoren (CDN) were performed. Efficacy of CDN-np in animals pre-immunized with HS-np (combined strategy) was explored and blood bacterial clearance determined. The CDN measured protein binding was 86.9%. CDN significantly increased phagocytosis in vitro,). In non pre-immunized animals, %TMIC values for CDN of <35% (total) and <19% (free) were associated with 100% survival. Significant differences in survival were found between HS-np alone (<20%) or CDN-np alone (<20%) vs. the combined strategy (90%, 60% and 60% for strains 1515, 69, and 48), with %TMIC (total/free) of 22.8%/14.3%, 26.8%/16.0%, and 22.4%/12.7% for strains 1515, 69, and 48, respectively. Prior to the second dose (8 h), median bacterial counts were significantly lower in animals surviving vs. dead, at day 7. A flow cytometry assay was used to determine whether complement-mediated immunity against three antibiotic-resistant S. pneumoniae clinical isolates is enhanced in the presence of subinhibitory concentrations of CDN and ceftriaxone (CRO). The binding of acute phase proteins, such as C-reactive protein and serum amyloid P component, and of complement component C1q to pneumococci was enhanced in the presence of serum plus either of the two antibiotics. Both antibiotics therefore trigger the activation of the classical complement pathway against S. pneumoniae. C3b deposition was also increased in the presence of specific anti-pneumococcal antibodies and subinhibitory concentrations of CDN and CRO confirming that the presence of these antibiotics enhances complement-mediated immunity to S. pneumoniae. To investigate whether LytA, the main cell wall hydrolase of S. pneumoniae, might be involved in this process, lytA mutants were constructed. In the presence of antibiotics, loss of LytA was not associated with enhanced C3b deposition on the pneumococcal surface, which confirms the importance of LytA in this interaction.
Using CDN and CRO to promote the binding of acute phase proteins and C1q to pneumococci, and to increase C3b deposition when anti-pneumococcal antibodies are present, might help reduce the impact of antibiotic resistance in S. pneumoniae infections. The results of this study offer new insights into the development of novel therapeutic strategies using some antibiotics by increasing the efficacy of the host immune response to efficiently recognize pneumococcal resistant strains. These findings reinforce the concept that immunoprotection to overcome resistance may provide lifesaving strategies. CONCLUSIONS: This study confirms that the LytB and LytC, two CWHs 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. In the same way, our results demonstrate that LytA and Ply are very important virulence factors of S. pneumoniae that divert complement-mediated immunity allowing the bacterium an efficient dissemination to the systemic circulation, and both proteins impair the recognition and phagocytosis of S. pneumoniae by professional phagocytes. In addition, the findings of the current Thesis demonstrate a novel role for PSGL-1 in host defence against pneumococcus. The impact of antibiotic resistance in S. pneumoniae infections might be reduced using cefditoren and ceftriaxone to promote the binding of acute phase proteins and C1q to pneumococci, and to increase C3b deposition, when anti-pneumococcal antibodies are present. Therefore, immunoprophylaxis approaches to diminish the impact of antibiotic resistance may be a novel and promising strategy to increase the outcome of pneumococcal infection.
Description191 p.-42 fig.-8 tab.-1 anexo
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