Refine
Year of publication
- 2022 (3)
Document Type
- Article (2)
- Doctoral Thesis (1)
Language
- English (3)
Has Fulltext
- yes (3)
Is part of the Bibliography
- no (3)
Keywords
- pneumococci (3) (remove)
Institute
Publisher
- John Wiley & Sons, Ltd (1)
- MDPI (1)
Abstract
Streptococcus pneumoniae infections are a leading cause of death worldwide. Bacterial membrane vesicles (MVs) are promising vaccine candidates because of the antigenic components of their parent microorganisms. Pneumococcal MVs exhibit low toxicity towards several cell lines, but their clinical translation requires a high yield and strong immunogenic effects without compromising immune cell viability. MVs are isolated during either the stationary phase (24 h) or death phase (48 h), and their yields, immunogenicity and cytotoxicity in human primary macrophages and dendritic cells have been investigated. Death‐phase vesicles showed higher yields than stationary‐phase vesicles. Both vesicle types displayed acceptable compatibility with primary immune cells and several cell lines. Both vesicle types showed comparable uptake and enhanced release of the inflammatory cytokines, tumor necrosis factor and interleukin‐6, from human primary immune cells. Proteomic analysis revealed similarities in vesicular immunogenic proteins such as pneumolysin, pneumococcal surface protein A, and IgA1 protease in both vesicle types, but stationary‐phase MVs showed significantly lower autolysin levels than death‐phase MVs. Although death‐phase vesicles produced higher yields, they lacked superiority to stationary‐phase vesicles as vaccine candidates owing to their similar antigenic protein cargo and comparable uptake into primary human immune cells.
Antibiotic resistance in pneumococci contributes to the high pneumococcal deaths in children. We assessed the molecular characteristics of multidrug-resistant (MDR) pneumococci isolated from healthy vaccinated children under five years of age in Cape Coast, Ghana. A total of 43 MDR isolates were selected from 151 pneumococcal strains obtained from nasopharyngeal carriage. All isolates were previously serotyped by multiplex PCR and Quellung reaction. Susceptibility testing was performed using either the E-test or disk diffusion method. Virulence and antibiotic resistance genes were identified by PCR. Molecular epidemiology was analyzed using multilocus sequence typing (MLST). Vaccine-serotypes 23F and 19F were predominant. The lytA and pavB virulence genes were present in all isolates, whiles 14–86% of the isolates carried pilus-islets 1 and 2, pcpA, and psrP genes. Penicillin, tetracycline, and cotrimoxazole resistance were evident in >90% of the isolates. The ermB, mefA, and tetM genes were detected in (n = 7, 16.3%), (n = 4, 9.3%) and (n = 43, 100%) of the isolates, respectively. However, >60% showed alteration in the pbp2b gene. MLST revealed five novel and six known sequence types (STs). ST156 (Spain9V-3) and ST802 were identified as international antibiotic-resistant clones. The emergence of international-MDR clones in Ghana requires continuous monitoring of the pneumococcus through a robust surveillance system.
Group A streptococcus (GAS) and Streptococcus pneumoniae are both Gram-positive bacteria that asymptomatically colonise various human body parts. Both microbes cause diseases ranging from mild to severe invasive infections. The later are associated with high mortality. GAS is the major microbial aetiology of type II necrotising skin and soft tissue infections (NSTIs). Type II NSTIs typically affect the lower and upper limbs of healthy young adults and often require debridement as a surgical intervention to prevent the spread of infection. S. pneumoniae is the major cause of respiratory tract infections including community-acquired pneumonia in young children and the elderly. Although most respiratory tract infections are successfully treated with antibiotics, emerging antibiotic resistance is a major cause of concern. Secreted virulence factors of Gram-positive bacteria play a major role in the successful invasion of host tissues causing different diseases. Additionally, they facilitate the spread of infection, contribute to tissue pathology, and potentially act as immune evasion mechanisms. This thesis summarises the consequences of streptococcal pyrogenic exotoxin B (SpeB), a potent cysteine protease secreted by GAS and pneumococci-derived hydrogen peroxide (H2O2) on host responses.
GAS have developed genetic or phenotypic ways of adapting to the immune response to escape immune clearance. Analysis of GAS clones recovered from NSTI patient biopsies exhibit a mixed SpeB phenotype, with most clones being SpeB negative. SpeB negative clones have been associated with hyper-virulence. In Paper II, we showed that SpeB negative GAS clones recovered from tissue exhibit reversible impaired SpeB secretion due to environmental factors. In addition, mutations in covS and ropB, the major transcriptional regulators of SpeB expression, were responsible for the irreversible loss of SpeB expression. Immunohistochemistry analysis demonstrated that neutrophil degranulation, necrosis and excessive inflammation observed in NSTIs patient biopsies correlated with bacterial load and SpeB negativity of clones. Proteomic data analysis showed that SpeB negative GAS recovered from neutrophil infection harboured the protease intracellularly suggesting that the bacteria expressed but did not secrete SpeB. We have also shown that neutrophil-derived reactive oxygen species, H2O2 and hypochlorous acid, drive the SpeB negative phenotype. The SpeB negative clones survived neutrophil-mediated antimicrobial killing and induced excessive degranulation when compared with SpeB positive clones. These results provide new insights into GAS fitness induced by host factors in tissue and may be useful for the development of new treatment strategies in NSTIs.
Pneumococci produce H2O2 as a by-product of carbohydrate metabolism in a reaction catalysed by pyruvate oxidase SpxB. However, very little is known about the effects of pneumococcal H2O2 as a virulence factor. Our study aimed to investigate the role of H2O2 in initiating epithelial cell death, focusing on apoptosis and pyroptosis. In Paper III, we showed that pneumococci-derived H2O2 caused epithelial cell cytotoxicity by priming and activating the NLRP3 inflammasome resulting in subsequent IL-1β production and release. Additionally, H2O2 caused apoptotic and pyroptotic cell death as evidenced by activation of caspase-3/7 and caspase-1, respectively. However, the release of IL-1β was dependent on apoptosis and not pyroptosis since inactive gasdermin D was detected post-infection. These observations were not detected in the absence of H2O2. Overall, we showed the damaging effects of pneumococci-derived H2O2 on human bronchial epithelial cells.