The genes encoding virulence-associated proteins and the capsule of Streptococcus pneumoniae are upregulated and differentially expressed in vivo

Abstract

The polysaccharide capsule of Streptococcus pneumoniae and several wellcharacterized virulence proteins are known to contribute to the pathogenesis of pneumococcal disease. However, there is a paucity of data on the expression of their respective genes in vivo. In this study, the relative abundance of the mRNA transcripts of the genes encoding pneumolysin (ply), pneumococcal surface protein A (pspA), pneumococcal surface antigen A (psaA) and cholinebinding protein A (cbpA), and of the first gene of the capsular polysaccharide biosynthesis locus (cps2A), was measured in virulent type 2 pneumococci harvested from the blood of BALB/c mice at 12 h and 24 h following intraperitoneal infection. The mRNA levels were then compared, using relative quantitative RT-PCR, with those present in organisms grown in serum broth. The expression of ply was upregulated threefold at 12 h, and 10-fold at 24 h post-infection ; the expression of pspA and psaA was upregulated threefold and fivefold, respectively, at 12 h post-infection. Interestingly, the expression of pspA was 36-fold higher at 24 h post-infection whereas the expression of cps2A was upregulated approximately fourfold at 12 and 24 h post-infection. However, cbpA mRNA levels remained comparable in vivo and in vitro. When organisms were grown in whole blood or THY broth, the relative expression of these genes in the two growth media also differed markedly. This work provides direct molecular evidence that known virulence-associated genes of S. pneumoniae are differentially expressed in vivo. Data on the relative expression of these genes in different growth media also suggests that the regulation of expression of these genes is highly complex and multifactorial.Keywords : pneumococcus, pneumolysin, surface proteins, mRNA, quantitative RT-PCR INTRODUCTIONStreptococcus pneumoniae is a very important human pathogen, causing a spectrum of diseases including pneumonia, meningitis, bacteraemia and otitis media (Musher, 1992 ;Tuomanen et al., 1995). The incidence of pneumococcal disease is particularly high in children under the age of 5 years, the elderly and immunocompromised individuals (Garenne et al., 1992 ; World Health Organization, 1997 (World Health Organization, 1997). Furthermore, the alarming rate at which this organism is acquiring resistance to multiple antimicrobials has exacerbated the high morbidity and mortality resulting from pneumococcal disease (Whitney et al., 2000). The polysaccharide (PS) capsule is strongly anti-phagocytic and is a sine qua non of pneumococcal virulence (Austrian, 1981). There are 90 recognized PS serotypes ; synthesis of the serotype-specific capsular PS is encoded by distinct clusters of up to 20 tightly linked genes transcribed as single operons (Paton & Morona, 2000). Antibodies to the PS are highly protective, and the global impact of pneumococcal disease has led to the development of polyvalent PS and PS-protein conjugate A. D. Ogunniyi, P. Giammarinaro and J. C. Paton vaccine formulations. Although appreciable levels of success h...

In vitro expression of the first capsule gene of Streptococcus pneumoniae, cpsA, is associated with serotype-specific colonization prevalence and invasiveness

“…Transcription of cpsA has been shown by RT-PCR to be upregulated fourfold in bacteria recovered 24 h after intraperitoneal infection compared to expression in vitro (Ogunniyi et al, 2002). In contrast, a microarray analysis found no increase in cpsA expression in bacteria from the blood of mice compared to bacteria grown in vitro (Orihuela et al, 2004), and no difference in cpsA expression was found among bacteria isolated from the nasopharynx, lungs and blood of mice infected intranasally (LeMessurier et al, 2006).…”

The two-component signal transduction system RR06/HK06 regulates expression of cbpA in Streptococcus pneumoniae

“…S. pneumoniae were grown in Todd Hewitt broth with 1% yeast extract (THY), serum broth (SB), or on blood agar, as described (2). Escherichia coli strains were grown in LB broth or LB agar (11).…”

“…We have demonstrated that several well characterized virulence genes of S. pneumoniae, including ply, pspA, psaA, and cbpA are differentially expressed under different environmental conditions (2). Of these genes, cbpA (also known as pspC) was of particular interest, because it is located only 750 nt downstream of rr06͞hk06, and a number of prokaryotic TCSTSs regulate adjacent genes on the chromosome, including the blpH͞R system from the pneumococcus (26).…”

“…Thus, the regulation of virulence factors in response to external stimuli may be a principal determinant of the success or failure of the infection. Recently, several virulence-associated genes of S. pneumoniae have been shown to be differentially expressed in varying in vitro and in vivo conditions, but the underlying mechanism is unknown (1,2). Two-component signal-transduction systems (TCSTSs) control the expression of virulence factors in a wide range of bacterial species in response to external stimuli (reviewed in ref.…”

Differential expression of key pneumococcal virulence genes in vivo

“…Lungs were removed and homogenized on ice in 2 ml sterile PBS using a tissue homogenizer (Cat X120, Germany). To separate pneumococci from host cells, lung homogenates and blood samples were centrifuged at 855 g for 6 min at 4 uC, as described previously (Ogunniyi et al, 2002). Nasopharyngeal washes, and lung and blood supernatants were subsequently centrifuged at 15 500 g for 2 min at 4 uC, and the bacterial pellet was stored at 280 uC until further processing.…”