Pathogenesis Of Infectious Disease Biology Essay

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The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms

Sanchez et al. (2010) article on Pneumococcal Serine-Rich Repeat Proteins (PsrP) gives an in depth study on the role of PsrP as an adhesin and the domains within its structure that facilitates its function. The article elucidates what has already been discovered on PsrP as an encoded pathogenicity island Streptococcus pneumoniae adhesin and has further added to the findings based on observed experimental results.

Invasive clones of S. pneumoniae that have the pathogenicity island psrP-secY2A2 carry PsrP, which is the largest bacterial protein known, and is a member of the serine-rich repeat protein (SRRP) family that are adhesins. PsrP consists of a cleavable N-terminal signal peptide, a short serine-rich repeat region (SRR1), a basic region (BR) also referred to as a unique non-repeat region (NR) composed of basic amino acids, which is followed by a second very long serine-rich repeat region (SRR2), and a C-terminal cell wall anchor domain containing an LPXTG motif. [1, 2, 3]

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Work conducted by Obert, Sublett et al. (2006) focusing on the genomic content of invasive S. pneumoniae serotypes showed that PsrP encoded in pathogenicity islands is necessary for virulence in mice. This was concluded from the observation that intranasal administration of PsrP-deficient mutants in mice caused slower bacterial entry into the bloodstream, and was less effective at killing mice compared to wild-types (WT). Thus, this demonstrated that PsrP is needed for efficient entry into the bloodstream of infected mice, presumably from the lungs. [4] Works by Rose et al. (2008) indirectly complemented Obert et al. findings and showed that PsrP-deficient mutants are unable to establish lung infection in mice by adhering to bronchial and alveolar [5]. Thus, it is evident that PsrP adhesin contributes to S. pneumoniae virulence.

Sanchez et al. (2010) article's background was partly based on the earlier work conducted by Shivshankar et al (2009). Shivshankar et al. expanded on the findings of Rose et al. whom demonstrated that PsrP adhesion is mediated by the amino terminus as antibodies against that domain is observed to inhibit adhesion in TIGR4; a S. pneumoniae serotype 4 strain containing psrP-secY2A2. They demonstrated that the PsrP ligand is restricted to the respiratory tract as PsrP deficient mutants T4ΔPsrP and T4ΔPsrP-secY2A2 were less effective at attaching to pneumocytes and bronchial epithelial cells compared to the wild type, but their binding to nasopharyngeal epithelial cells were not affected. As capsular polysaccharides have been shown to inhibit pneumococcal adhesion (Kim, Romero-Steiner et al. 1999), and as TIGR4 is encapsulated, Rose et al. suggested that the BR domain is extended beyond the capsular polysaccharide by the extremely long SRR2 domain in order to mediate lung cell adhesion. Lastly they determined that passive immunization with PsrP anti-serum protected mice against pneumococcal challenges, as it inhibited TIGR4 from adhering to lung cells in vitro and reduces the amount of bacteria in the lungs and blood of challenged mice. [5]

The works of Shivshankar et al. demonstrated the presence of psrP-secY2A2 in invasive globally distributed S. pneumoniae clones and serotypes that the current conjugate vaccine does not protect against, by searching the genomes of isolates that have already been sequenced and are known to cause mortality world-wide. They determined homologues of TIGR4 psrP-secY2A2 genes in 6 out of the 19 genomes examined, and that 5 out of the 13 serotypes containing the pathogenicity island are not covered by the conjugate vaccine. Also through immunofluorescent microscopy and Periodic Acid-Schiff stain test they showed that PsrP is present on the bacteria surface and that PsrP is glycosylated respectively. [2]

Shivshankar et al. also expanded on the work of Rose et al. and determined the particular region of the BR domain A.As 273-341 that is responsible for adhesion and the ligand to which it binds to, K10 on lung cells. This was tested by using recombinant PsrP (rPsrP) constructs containing A.A 273-341 which was observed to bind to lungs cells 8-20 fold greater than the control and other sections of PsrP. As for K10 on lung cells it was identified as the ligand for PsrP by passing A549 cells; a human type II pneumocyte cell line over a column with rPsrPBR in column chromatography. The eluant was separated, stained, and analysed by MALDI-TOF, identifying K10 as the bound protein. The PsrP-K10 interaction was subsequently confirmed by immunoprecipitation, ELISA and by immunofluorescence. Flow cytometry was used to detect K10 on the surface of A549 and on murine LA-4 respiratory epithelial cells. It was also observed that changes in K10 levels modulated PsrP mediated adherence in S. pneumoniae; as silencing K10 in A549 cells reduced the level of bacteria binding in contrast to increasing K10 expression which increased pneumococcal attachment. Changes in K10 expression had no affect on the adhesion of encapsulated S. pneumoniae PsrP deficient mutant T4ΩpsrP, thereby indicating its specificity for PsrP. It was further tested whether K10 was present on Detroit 562 nasopharyngeal cells, using western blotting, ELISA, immunofluorescent imaging and cytometric analyses all of which gave negative results. Thus this explained why PsrP mutants are able to colonise the nasopharynx normally as observed by Rose et al. and why rPsrP with A.A 273-341 failed to bind, as the absence of K10 prevents PsrP mediated attachment. [2]

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They also confirmed the hypothetical model of Rose et al. that PsrP adhesion depends on SRR2 length, by creating a plasmid that encodes for a truncated PsrP with just 33 SRRs in the SRR2 region (PsrP SRR2(33)) and another PsrP that lacks the BR domain (PsrP SRR2(33)-BR). It was observed upon complementing PsrPSRR2(33) with T4ΩpsrP (an encapsulated derivative of TIGR4 that lacks PsrP) that T4ΩpsrP failed to adhere to A549 cells. In contrast the complementation of PsrPSRR2(33) with T4RΩpsrP (an un-encapsulated derivative of TIGR4 that lacks PsrP) restored A549 attachment. Of note, the complementation of PsrPSRR2(33)-BR with T4RΩpsrP had no adhesion effect. Thus, these observations indicate that the BR domain is necessary for PsrP adhesion, and that an extended SRR2 domain is required for capsulated S. pneumoniae attachment, as it seems to extend the BR domain through the capsular polysaccharide. [2]

Lastly they showed that active immunization of mice with rPsrP BR domains also confers protection against pneumococcal challenges, as significant bacteria decrease was observed in the blood as well as a reduced mortality rate. [2]

Sanchez et al. work on S. pneumoniae PsrP is the most recent and up to date. The scientific background that forms the basis of their work is well established, and their work merely advances on it. The significant advances made in their work are:

They are the first to show that biofilm structures are formed in the lungs of animals infected with S. pneumoniae [1]. Biofilms are organised aggregates of bacteria cells embedded in an extra-cellular matrix of exopolysaccharides. The formation of biofilms caters for bacteria growth and survival in hostile environments. Biofilms have been associated with more than 60% of human bacterial infections, being able to resist antibiotic agents and host immunity. It has been a notable observation that cells that aggregate and form communities tend to have greater resistance to antibiotics, up to 1000 times more resistant than the same cells that are planktonic (free-swimming and not attached to any surface). [6, 7]

Biofilm formation has been related with chronic infections, one of which is chronic otitis caused by S. pneumoniae biofilms in the middle ear of children, so it is known that S. pneumoniae forms biofilms but not until recently that they also form them in the lungs mediated by PsrP. Sanchez et al. tested for this phenomenon in in-vivo mice, by infecting mice with TIGR4 and its isogenic T4ΔpsrP (PsrP deficient mutant). It was observed through examining the lung section of mice using scanning electron microscopy (SEM) that the bronchial and alveolar epithelial cells had large aggregate clusters of TIGR4, which were not seen in the lungs of T4ΔpsrP infected mice (see Figure 1). Two days after, mice fluid from the nasal and bronchoalveolar lavage was collected for quantitative analysis and examined after being gram-stained under a microscope. In both fluids TIGR4 had significantly greater bacterial aggregation, forming the largest aggregates composed of > 100 bacteria. These observations indicate that PsrP is involved in in-vivo biofilm formation in the lungs and nasopharynx, despite previous studies showing PsrP was not required for nasopharynx colonization. [1, 7]

Figure (1) - SEM images of the trachea and alveoli illustrating the formation of biofilms in wild type and PsrP deficient mice [copied from [1]]

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As the BR domain is responsible for the binding of SRRPs to host cells, Sanchez et al. sought to determine whether the BR domain is also responsible for the observed intra-species bacterial interactions involved in biofilm formation. To test this they used S. pneumoniae PsrP deficient mutants; T4ΩpsrP (encapsulated) and T4RΩpsrP (unencapsulated) that either expressed PsrPSRR2(33), or PsrPSRR2(33)-BR, or carried an empty expression vector pNE1. The strains were then tested on their ability to form biofilms in silicone lines. When T4ΩpsrP and T4RΩpsrP was complemented with PsrPSRR2(33) partial aggregation was observed in the silicone lines under a microscope. However no aggregation was observed when either T4ΩpsrP or T4RΩpsrP was complemented with PsrPSRR2(33)-BR. This suggests that the BR domain plays a role in aggregation. [1]

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They further determined the section of the BR domain responsible for bacteria-bacteria interactions, by using His-tagged BR constructs (Figure 2), labelled with Cy3 and purified from E. coli, they attempted to bind them to TIGR4 and T4ΔpsrP. It was observed that only the full-length rBR and rBR.A was able to interact with TIGR4 but not with T4ΔpsrP. This showed them that the BR domain needs to bind to a PsrP to form aggregation and the portion of the BR domain responsible is located within A.A 122-166, the section not shared between rBR.A and rBR.B. [1]

Figure (2) - Recombinant PsrP constructs [copied from [1]]

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Subsequently, they tried to determine the actual part of the PsrP that the BR binding domain binds, by using Far Western blotting and co-immunoprecipitation experiments they showed that the Gst-BR only had affinity for the full length rBR or rBR.A, thus it was determined that bacteria aggregation is mediated through BR-BR interactions, with the A.As 122-166 being the binding region. [1]

It is already known through the works of Shivshankar et al. and Rose et al that through active and passive immunization, antibodies against the BR domain (A.A 273-341) can inhibit S. pneumoniae from binding to lung cells. Sanchez et al. further sought out to discover whether antibodies against BR can also inhibit bacterial aggregation in the biofilm line model. To do this they made a 1:1000 dilution of antiserum against the BR domain in Todd Hewitt Broth (THB) and observed microscopically whether bacterial aggregates formed. They noticed bacterial aggregation was inhibited in the biofilm line model. They further tested whether the BR antiserum had any effect on TIGR4 biofilm formation, so they tested it against unrelated clinical isolates. They found that biofilm formation was inhibited in two unrelated clinical isolates, but there was an invasive serotype 14 isolate that lacked a PsrP, and BR antiserum was unable to prevent it from forming biofilm. This shows that biofilms can still be formed independent of PsrP. [1]

The last significant advancement made by Sanchez et al. was through demonstrating that other SRRPs such as gspB and sraP of S. gordonii and S. aureus respectively also promote bacterial aggregation, thereby discovering an unrecognized role for members of the SRRP family. Thus SRRPs have dual roles one as a host and another as a bacterial adhesin. [1]

To summarize the results of Sanchez et al. work they clearly illustrate through methods that involved the use of S. pneumoniae bacterial strains from wild-types to mutants that were diluted and cultured in mice and models, and further analysed and examined through the use of a range of methods such as scanning electron microscopy (SEM) and fluorescent microscopy that PsrP promotes bacterial intra-species adhesion in the lungs and nasopharynx. And through the use of other analytical methods such as Far western analysis of BR interactions, co-immunoprecipitation with rBR, and from the use of competitive peptides and antibodies against BR they further reached the conclusions that it is the BR domain of the PsrP, particularly the region A.A 122-166 that is responsible for mediating and promoting the formation of biofilms through bacterial interactions, and that antibodies against the BR domain inhibited this observation. [1]

The results of Sanchez et al. findings may be significant for the microbial pathogenesis of S. pneumoniae, however how the full implications of their findings on PsrP interactions contributes to the pathogenesis of S. pneumoniae needs to be further determined. But generally it is known through other microbes that form biofilms that they are able to inhibit host cell defences such as phagocytoses and can also protect against defensins, and can reduce antibiotic susceptibility through the biofilm matrix impeding the rate of antibiotic penetration giving time for the expression of genes within the biofilm that mediates resistance. Biofilms are also formed during numerous chronic infections, and their formation is simply a strategy that caters for microbial survival, acting as a reservoir for the pathogens by providing a stable protective environment from which they can disseminate from and find new surfaces, thus during the early stages of disease biofilms serves as a focal point of infection. [1, 6, 7] However, the actual affects biofilm formation in the lungs and nasopharynx has on S. pneumoniae pathogenesis, if any, still needs to be determined.

From the work and findings of Sanchez et al. further studies in the future can be conducted to determine what is still unknown, such as identifying the specific A.As responsible for the adhesive properties of the BR domain, as only the region of the BR domain that mediates adhesion is known, but not the particular A.As within the region. Also determine the protein structure of the BR domain, and observe how the sub-domains interact with K10 on lung cells and PsrP on other pneumococci. And to determine whether antibodies can neutralize bacterial aggregation in vivo as it has been observed to do so within the biofilm line model. Lastly the full extent as to how PsrP interactions from the binding of lung cells to forming biofilms contributes to the pathogenesis of S. pneumoniae needs to be studied. [1]