Adaptation of the Staphylococcus aureus leukocidin LukGH for the rabbit host by protein engineering.

Host defense against Staphylococcus aureus greatly depends on bacterial clearance by phagocytic cells. LukGH (or LukAB) is the most potent staphylococcal leukocidin towards human phagocytes in vitro, but its role in pathogenesis is obscured by the lack of suitable small animal models because LukGH has limited or no cytotoxicity towards rodent and rabbit compared with human polymorphonuclear cells (PMNs) likely due to an impaired interaction with its cellular receptor, CD11b. We aimed at adapting LukGH for the rabbit host by improving binding to the rabbit homolog of CD11b, specifically its I-domain (CD11b-I). Targeted amino acid substitutions were introduced into the LukH polypeptide to map its receptor interaction site(s). We found that the binding affinity of LukGH variants to the human and rabbit CD11b-I correlated well with their PMN cytotoxicity. Importantly, we identified LukGH variants with significantly improved cytotoxicity towards rabbit PMNs, when expressed recombinantly (10-15-fold) or by engineered S. aureus strains. These findings support the development of small animal models of S. aureus infection with the potential for demonstrating the importance of LukGH in pathogenesis.

Age-related changes in the levels and kinetics of pulmonary cytokine and chemokine responses to Streptococcuspneumoniae in mouse pneumonia models.

Streptococcuspneumoniae is a major human pathogen at the extremes of age. The elderly are particularly vulnerable to S.pneumoniae, the most common causative agent of bacterial pneumonia in this population. Despite the availability of vaccines and antibiotics, mortality rates associated with pneumococcal pneumonia in this age group remain high. In light of globally increasing life-expectancy, a better understanding of the patho-mechanisms of elderly pneumococcal pneumonia, including alterations in innate immune responses, is needed to develop improved therapies. In this study we aimed at investigating how increased susceptibility to pneumococcal infection relates to inflammation kinetics in the aged mouse pneumonia model by determining pulmonary cytokine and chemokine levels and comparing these parameters to those measured in young adult mice. Firstly, we detected overall higher pulmonary cytokine and chemokine levels in aged mice. However, upon induction of pneumococcal pneumonia in aged mice, delayed production of certain analytes, such as IFN-γ, MIG (CXCL9), IP-10 (CXCL10), MCP-1 (CCL2), TARC (CCL17) and MDC (CCL22) became apparent. In addition, aged mice were unable to control excess inflammatory responses: while young mice showed peak inflammatory responses at 20 h and subsequent resolution by 48 h post intranasal challenge, in aged mice increasing cytokine and chemokine levels were measured. These findings highlight the importance of considering multiple time points when delineating inflammatory responses to S.pneumoniae in an age-related context. Finally, correlation between pulmonary bacterial burden and cytokine or chemokine levels in young mice suggested that appropriately controlled inflammatory responses support the host to fight pneumococcal infection.

Assessing the function of pneumococcal neuraminidases NanA, NanB and NanC in in vitro and in vivo lung infection models using monoclonal antibodies.

Streptococcus pneumoniae isolates express up to three neuraminidases (sialidases), NanA, NanB and NanC, all of which cleave the terminal sialic acid of glycan-structures that decorate host cell surfaces. Most research has focused on the role of NanA with limited investigations evaluating the roles of all three neuraminidases in host-pathogen interactions. We generated two highly potent monoclonal antibodies (mAbs), one that blocks the enzymatic activity of NanA and one cross-neutralizing NanB and NanC. Total neuraminidase activity of clinical S. pneumoniae isolates could be inhibited by this mAb combination in enzymatic assays. To detect desialylation of cell surfaces by pneumococcal neuraminidases, primary human tracheal/bronchial mucocilial epithelial tissues were infected with S. pneumoniae and stained with peanut lectin. Simultaneous targeting of the neuraminidases was required to prevent desialylation, suggesting that inhibition of NanA alone is not sufficient to preserve terminal lung glycans. Importantly, we also found that all three neuraminidases increased the interaction of S. pneumoniae with human airway epithelial cells. Lectin-staining of lung tissues of mice pre-treated with mAbs before intranasal challenge with S. pneumoniae confirmed that both anti-NanA and anti-NanBC mAbs were required to effectively block desialylation of the respiratory epithelium in vivo. Despite this, no effect on survival, reduction in pulmonary bacterial load, or significant changes in cytokine responses were observed. This suggests that neuraminidases have no pivotal role in this murine pneumonia model that is induced by high bacterial challenge inocula and does not progress from colonization as it happens in the human host.

Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.