Is lactate an undiscovered pneumococcal virulence factor?

Is lactate an undiscovered pneumococcal virulence factor?

Streptococcus pneumoniae is Gram-positive alpha haemolytic bacteria that commonly found in the nosphrynax of elderly people and young children, it causes approximately 2 million deaths mostly children under age of 5 year and people over 60 years of age. Most important diseases caused by S. pneumoniae are including pneumonaie, meningitis and bacteraemia. The pathogens can be transmitted through contact. Streptococcus pneumoniae obtains its energy mainly carbohydrates through fermentation process. However, in some situations where there are limited sugars or in the presence of galactose the homolactic fermentation is shifted to mixed fermentation in which in addition to lactate, ethanol, formate and acetate are formed. In this study, the role of lactate (lactic acid) and formate (formic acid) in bacterial competition and cytotoxicy was investigated. We hypothesised that lactic acid and formic acid are able to contribute to the virulence of streptococcus pneumoniae. Bacteria were grown on either BHI or BAB. The killing assay was done by exposing various acids on S.pneumoniae as control then lactic acid producing bacteria and non-acid producing bacteria was tested with these acids. Growth assay experiment was done followed by cytotoxicity test using A549 epithelial cells incubated for 24h. The effect of lactic acid for killing assay was significant. Similar effect was seen when lactic acid was exposed to A549 cells. However, a hydrochloric acid acid was unable to inhibit the growth of bacteria. This study concludes that lactic acid produced by Streptococcus pneumoniae is a potential virulence factor and may contribute to Streptococcus invasive disease.

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Chapter 1 Introduction

1.1 The biology of Streptococcus pneumoniae

Streptococcus pneumoniae is a normal inhabitant of human nasopharynx, and it is a member of lactic acid family that gets its energy mainly by the process of fermentation. It is Gram positive, and catalase negative. Under light microscope S. pneumoniae can be seen in pairs and short chains. In blood agar they can be seen as α-haemolytic.

S. pneumoniae is a fastidious facultative anaerobe that requires highly nutritious medium for growth. It grows in Brain Heart Infusion (BHI) media, as well as Blood Agar Base (BAB). This bacterium also grows in chemically defined medium that contains nutrients, such as vitamins, glucose, amino acids (Table 1) and pyruvate. However, the most easily observable characteristic of S. pneumoniae is its sensitivity to optochin (ethylhydrocupreine). This makes the pneumococcus distinguishable from other alpha haemolytic streptococcus. Like other Gram positive bacteria S. pneuminae possess three major surface layer that can be distinguishable: cell wall, plasma membrane and capsule (Alonsodevelasco, E. et, al 1995). There are more than 90 serotypes of S.pneumoniae based on their capsular polysaccharides coats.

1.2 The Genome:

The complete genome sequence of a type 4 isolate of S. pneumoniae comprises a single circular chromosome of 2,160,837 base pairs (bp) about 40% of G+C content(Tettelin et al., 2001). This genome contains 2236 predicted coding regions; of these genes around 64% are assigned a biological role (Tettelin et al., 2001). It also contains 73 noncoding RNA genes that include four rRNA operons. Moreover, S. pneumoniae has a high capacity for DNA uptake (Hoskins, J. et al 2001).

The pneumococcal genomes contain a considerable number of insertion elements such as transposon remnants and repeat sequences. The large number of insertion elements in the genome indicates that the pneumococcal genome is exposed to common inter and intra-genomic events. ( Lanie.J.A.,et, al. 2006)

1.3 The diseases caused by S. pneumoniae and their epidemiology:

The diseases caused by the pneumococcus is life threatening and include pneumonia, meningitis, bacteraemia and septicaemia. Additionally, it also causes otitis media, sinusitis, osteomyelitis, and peritonitis. The microorganism is also responsible for endocarditis, and septic arthritis (Kilian, 2007). The diseases caused by S.pneumoniae are results from either direct extension from the nasopharynx or by invasion and haematogenous spread.

Pneumonia is a very important cause of mortality and morbidity amongst elderly people.

( Nagaoka, S. et al..). Despite the availability of pneumococcal vaccine this microorganism still pose a great challenge to any attempt to eradicate or limit the spread of the disease because rising antibiotic resistance and limitations of vaccines. The pneumococcal infections are responsible for more than 1.6 million deaths each year worldwide (WHO, 2008). The highest incident of this disease occurs in children under the age of 5 year and in the elderly. Also very high incident in patients with predisposing conditions such as asplenia and those who are immune-compromised are reported.

1.4 The pneumococcal virulence determinants

1.4.1 Capsule: S.pneumoniae possess polysaccharide capsule which is considered as the most important virulence factor, because unencapsulated pneumococcus is almost harmless while the encapsulated bacteria from the same species cause disease (Alonsodevelasco,et,al 1995). It has been found that encapsulated strains are approximately 105 more virulent than unencapsulated strains (Alonsodevelasco,et,al 1995) I general, the vast majority of Streptococcus serotypes are unable to produce potential virulence (Lysenko, et al. 2010). The survival of the serotype in the blood stream and ability to cause invasive disease are mainly determined by the chemical structure of capsule polysaccharide and thicknes of capsule (Alonsodevelasco,et al. 1995).

1.4.2 Protein virulence determinants: Recent studies discovered that there are proteins that also contribute to virulence. They include, but are not limited to, hyaluronate lyase,(berry, M. et al., 1994), pneumolysin (Paton, J.c., et al 1986), neuraminidases (Elizabeth A. et., al 2002), galactosidases (Terra et al., 2010) and pyruvate formate lyase (Yesilkaya et al., 2009).

1.4.2a Hyaluronate lyase: Hyaluronate lyase degrades the hyaluronan, which is a hyaluronic acid derivative and its one of the most important polysaccharide component of animals, into disaccharide as a final product (Songlin, et al., 2000). Study carried by Berry et al. (1994), suggests that hyaluronidase plays vital role in migration of streptococci between tissues, in particular translocation from the lungs to vascular system. The other way in which hyaluronidase contributes the streptococcal pathogenesis is by allowing huge number of microorganisms to host tissue for colonization (Berry et al. (1994).

1.4.2b Pneumolysin: This is a membrane damaging toxin which inhibits neutrophil chemotaxis, phagocytosis and respiratory burst (Greenwood, D. et, al 2007). The sulfhydryl-activated cytolysin toxin functions by binding to cholesterol in host cell membranes. (Paton, J.c., et al 1986). It also damages blood vessels in the lungs and therefore, causes bleeding into air spaces. Moreover, pneumolysin leads to the activation of the classical complement pathway and the depletion of serum opsonic activity (Lock, R.A., 1988)

1.4.2c Neuraminidases: This enzyme is able to cleave N-acetylneuraminic acid from glycoproteins, such as mucin, oligosaccharides, and glycolipids on host cell surfaces. S. pneumoniae expresses several distinct neuraminidases. Studies carried out by Elizabeth A. et., al (2002) has indicated that neuraminidase activity might promote the colonization by decreasing the viscosity of mucus(Tong et al, 2000). The two neuraminidases (NanA and NanB) are part of virulence factors that cause disease (Tong et al, 2000). Although there are three forms of neuraminidases, NanA, NanB and NanC , the most abundant neuraminidase and probably the most important one is the Neuraminidase A(NanA ). Almost all the S. pneumoniae that were investigated has shown to have neuraminidase activity (Anirban et al., 2010). These investigations showed that NanA contributed to the colonisation of pneumococcus in the nasopharynax and al…………………………………….