Abstract
Streptococcus pneumoniae displays increased resistance to antibiotic therapy following biofilm formation. A genome-wide search revealed that SP 0320 and SP 0675 (respectively annotated as 5-keto-D-gluconate-5-reductase and glucose dehydrogenase) contain the highest degree of homology to CsgA of Myxococcus xanthus, a signaling factor that promotes cell aggregation and biofilm formation. Single and double SP 0320 and SP 0675 knockout mutants were created in strain BS72; however, no differences were observed in the biofilm-forming phenotypes of mutants compared to the wild type strain. Using the chinchilla model of otitis media and invasive disease, all three mutants exhibited greatly increased virulence compared to the wild type strain (increased pus formation, tympanic membrane rupture, mortality rates). The SP 0320 gene is located in an operon with SP 0317, SP 0318 and SP 0319, which we bioinformatically annotated as being part of the Entner-Doudoroff pathway. Deletion of SP 0317 also resulted in increased mortality in chinchillas; however, mutations in SP 0318 and SP 0319 did not alter the virulence of bacteria compared to the wild type strain. Complementing the SP 0317, SP 0320 and SP 0675 mutant strains reversed the virulence phenotype. We prepared recombinant SP 0317, SP 0318, SP 0320 and SP 0675 proteins and confirmed their functions. These data reveal that disruption of genes involved in the degradation of ketogluconate, the Entner-Doudoroff pathway, and glucose dehydrogenase significantly increase the virulence of bacteria in vivo; two hypothetical models involving virulence triggered by reduced in carbon-flux through the glycolytic pathways are presented.
Highlights
Streptococcus pneumoniae colonizes the human nasopharynx [1], and is an upper respiratory tract pathogen that is often the causative agent of both acute and chronic otitis media with effusion [2,3,4,5,6]
We shall refer to the SP 0317, SP 0318, SP 0319 and SP 0320 genes collectively as the kdg region as we bioinformatically annotated them as being involved in ketogluconate metabolism with some of the proteins likely part of the Entner-Doudoroff pathway, which is an alternative pathway to glycolysis in the catabolism of hexose to pyruvate [44, 45] (Fig 2)
Our results indicated that the membrane protein extract of the SP 0317 mutant strain had a 1.7 fold greater glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity compared to the wild type strain, whereas the membrane protein extract of the SP 0320 and SP 0675 mutant strains were similar to the wild type strain (1.1 and 1.2 fold greater activities compared to the wild type strain, respectively) (Fig 8)
Summary
Streptococcus pneumoniae (pneumococcus) colonizes the human nasopharynx [1], and is an upper respiratory tract pathogen that is often the causative agent of both acute and chronic otitis media with effusion [2,3,4,5,6]. The pneumococcus can cause life-threatening invasive diseases including pneumonia, bacteremia and meningitis that affect millions of persons annually throughout the world [9,10,11,12,13,14]. Infections, both acute and chronic, localized and invasive, caused by S. pneumoniae have become increasingly difficult to treat, due to the acquisition of genetic determinants rendering it antibiotic resistance and its ability to form a biofilm that is metabolically recalcitrant to antibiotic therapy [15,16,17,18,19]. The increase in number of bacterial cells during growth and aggregation allows for the more closely packed cells to promote C-signaling [23]
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