|Year : 2017 | Volume
| Issue : 4 | Page : 511-517
Distribution of different genes responsible for invasive characteristics, detection of point mutations in capsular gene wchA and biofilm production among the invasive and non-invasive isolates of Streptococcus pneumoniae
James John1, Kripa Shanker Kasudhan2, Reba Kanungo3, Savitri Sharma4, Vaishali Dohe5, K Prashanth1
1 Department of Biotechnology, School of Life Sciences, Pondicherry University, Puducherry, India
2 Department of Biotechnology, School of Life Sciences, Pondicherry University, Puducherry; Department of Pharmacology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry, India
4 Jhaveri Microbiology Centre, L V Prasad Eye Institute, Hyderabad, Telangana, India
5 Department of Microbiology, Byramjee Jeejeebhoy Government Medical College, Pune, Maharashtra, India
|Date of Web Publication||1-Feb-2018|
Dr. K Prashanth
Department of Biotechnology, School of Life Sciences, Pondicherry University, R. Venkataraman Nagar, Kalapet, Puducherry - 605 014
Source of Support: None, Conflict of Interest: None
Background: Streptococcus pneumoniae continues to cause morbidity and mortality across the globe, with developing countries bearing the brunt of the disease. It is mainly responsible for meningitis, pneumonia and septicaemia primarily in children, elderly and immunocompromised persons. Colonisation and persistence in the human nasopharynx occur during early childhood, and it appears to be prerequisite for invasive pneumococcal disease (IPD). Factors that help in persistent colonisation and subsequent invasion are ill understood. Several virulence factors have been incriminated for nasopharyngeal carriage (NC) as well as for the manifestation of the pathogenesis of IPD. Materials and Methods: This study attempts to characterise the S. pneumoniae isolates through analysing the distribution of different virulence markers such as lytA, ply, pbpA, eno, psaA, amiA, ciaR and wchA among the isolates obtained from disease and NC. A total of 37 isolates which include 14 invasive and 23 non-invasive isolates were investigated by polymerase chain reaction to detect the genes. Eight representative isolates were investigated for mutations in wchA by DNA sequencing that may responsible for capsular variation. Results: Ply, pbpA, amiA and eno were observed in a greater percentage of invasive isolates than non-invasive isolates though these differences are not statistically significant. Other two genes ciaH and psaA did not show any significant difference between two groups of isolates. Biofilm production was significantly higher in than non-invasive isolates when compared to invasive isolates. Sequence analysis of wchA revealed three significant point mutations or single-nucleotide polymorphisms (SNPs) among the isolates of one particular cluster (cluster III). These SNPs are responsible for a non-synonymous mutation in wchA bringing in an amino acid change in WchA protein, which is a part of the capsule of S. pneumoniae. Notably, all the three isolates present in cluster III had these SNPs and all of them were isolated from ocular infections. Conclusion: The results of our study implies a possible capsular variations among the isolates and this may have an impact on capsular typing.
Keywords: Autolysin, pneumolysin, Streptococcus pneumoniae, virulence genes, wchA point mutations
|How to cite this article:|
John J, Kasudhan KS, Kanungo R, Sharma S, Dohe V, Prashanth K. Distribution of different genes responsible for invasive characteristics, detection of point mutations in capsular gene wchA and biofilm production among the invasive and non-invasive isolates of Streptococcus pneumoniae. Indian J Med Microbiol 2017;35:511-7
|How to cite this URL:|
John J, Kasudhan KS, Kanungo R, Sharma S, Dohe V, Prashanth K. Distribution of different genes responsible for invasive characteristics, detection of point mutations in capsular gene wchA and biofilm production among the invasive and non-invasive isolates of Streptococcus pneumoniae. Indian J Med Microbiol [serial online] 2017 [cited 2019 Dec 16];35:511-7. Available from: http://www.ijmm.org/text.asp?2017/35/4/511/224425
| ~ Introduction|| |
Streptococcus pneumoniae continues to cause increased morbidity and mortality due to invasive infection in the vulnerable population. The problem is compounded by colonisation of this organism in the upper respiratory tract of asymptomatic carriers., Several factors such as capsular polysaccharides and other virulence factors can elicit and amplify the immune response when the S. pneumoniae undergoes a transition from coloniser to invasive ones.,,, Molecular mechanisms underlying the distinct central nervous system tropism of S. pneumoniae are not well characterised. Till date, a number of different virulence factors have been found in S. pneumoniae, each of them has different bioactivity and the presence of these factors varies with the kind and nature of disease severity., Emerging reports suggest that non-typable S. pneumoniae is typically associated with nasopharyngeal carriage (NC) and conjunctivitis. The genetic basis of the non-typability has been attributed to distinct point mutations in the wchA gene (a glycosyltransferase) leading to expression of truncated Wch protein. Investigations on the prevalence of different virulence genes among invasive and non-invasive isolates of S. pneumoniae may help in presumptive identification of disease-specific virulence factors involved in pathogenicity and understand the role of these factors among invasive and non-invasive isolates of S. pneumoniae. In the present study, we attempted to characterise the S. pneumoniae isolates through analysing the distribution of different virulence markers such as lytA, ply, pbpA, eno, psaA, amiA and ciaR. Further, capsular gene wchA was investigated for point mutations that may cause capsular variation.
| ~ Materials and Methods|| |
The study included a total of 37 S. pneumoniae isolates from Pondicherry Institute of Medical Science Hospital, Puducherry, L. V. Prasad Eye Institute, Bhubaneswar and B. J. Medical College, Pune, isolated from different clinical specimens and nasopharyngeal carriers of healthy volunteers [Table 1], during the period from March 2012 to December 2013. The clinical isolates were obtained from patients who are suffering mainly from invasive diseases such as pneumonia, bacteraemia, meningitis, ocular infection and bloodstream infections. The patient age range was from 1 to 72 years. Isolation of S. pneumoniae was from different clinical specimens such as blood, cerebrospinal fluid, bronchoalveolar lavage, sputum and corneal scrapings. S. pneumoniae ATCC49619 reference strain was used as a control for all the phenotypic tests. All the isolates were characterised to species level using simple phenotype tests such as Gram staining, optochin solubility and bile solubility assays. All the isolates were subjected to antimicrobial susceptibility by disc diffusion assay for clinically relevant antibiotics. The isolates were classified into two groups, namely, invasive and non-invasive isolates based on their origin. Fourteen isolates were from clinical samples obtained from patients with invasive infections such as meningitis, septicaemia and pneumonia. Twenty-three remaining isolates were grouped into a non-invasive group that were isolated from either ocular infections (n = 8) or from nasopharyngeal carriers in the paediatric group (n = 15).
|Table 1: Details of isolates, site of isolation, diagnosis and the polymerase chain reaction results of virulence factors of all the isolates of Streptococcus pneumoniae investigated in the study|
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Genomic DNA isolation
Genomic DNA isolation was performed for all isolates before polymerase chain reaction (PCR). Overnight bacterial broth culture of 2 ml was pelleted by centrifugation for 2 min at 12,000 g, and pellet was used for bacterial DNA isolation. Bacterial DNA was isolated using Qiagen (Hilden, Germany) bacterial genomic isolation kit as per manufacturer instruction. Isolated genomic DNA was visualised using 1% agarose gel and stained with ethidium bromide. The presence of different virulence genes was detected by the PCR amplification performed using thermal cycler (Veriti, Applied Biosystems). All the primers for virulence genes were designed in house by GeneTool software using the information of DNA sequences of genes that are available in the public domain (GenBank). The PCR primers and amplification conditions for PCR used in the study were given in [Table 2]. Amplified DNA was run on 1% agarose gel and visualisation under Bio-Rad Gel Doc.
All the isolates were screened by PCR for detection and distribution of virulence genes such as lytA that codes for autolysin, ply that codes for pneumolysin toxin, pbpA that codes for penicillin-binding protein 1A, eno thatcodes for anchorless surface protein enolase, ciaR regulatory gene and genes such as psaA and amiA that encode for adhesion proteins. Further, PCR was also used detect wchA that is implicated for capsule variation. All amplified wchA amplicons from different isolates were custom sequenced by Macrogen (South Korea). All the sequences of wchA were analysed for point mutations after performing multiple alignments with all the available DNA sequences of wchA genes in GenBank using ClustalW. All the isolates were tested for the biofilm-forming capabilities, and biofilm production abilities were later compared among the isolates of different origins. Biofilm assay was done using 96-well microtitre plates, and the optical density (OD) measurements for each of the wells were read at 595 nm (Bio-Rad 550 plate reader). The results and its interpretation were according to the procedure described elsewhere.
We compared the differences in the presence of virulence genes among the S. pneumoniae isolates obtained from invasive infections and with the isolates obtained from non-invasive infections/nasopharyngeal carriers and investigated for the significant association of virulence factors with a particular group or milieu. Contingency tables were calculated with Pearson's test or Fisher's exact test by comparing the proportions, wherever necessary. Differences were significant if the P value associated with the test was <0.05. Quantitative analysis for biofilm production by the two groups was performed by Pearson's correlation test.
| ~ Results|| |
All the isolates harboured lytA gene irrespective of the source of isolation. Invasive isolates showed the presence of ply (79%) and pbpA (57%) genes which were comparatively more than non-invasive isolates Ply (52%) and pbpA (30%). Similarly, amiA (71%) and eno (71%) genes were comparatively more in invasive isolates than the non-invasive ones amiA (61%) and eno (57%). CiaH and psaA positivity did not show any significant difference between these two groups [Figure 1]. Differences in the presence of virulence genes among the S. pneumoniae isolates obtained from invasive infections and with the isolates obtained from non-invasive infections/nasopharyngeal carriers were found to be statistically insignificant. However, this result could be due to a fewer number of isolates analysed in each group.
|Figure 1: Percentage distribution of various virulence factors among the isolates of Streptococcus pneumoniae|
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Quantitative analysis for biofilm production showed non-invasive isolates obtained from ocular infections, and nasopharyngeal carriers were producing significantly more amount biofilms (OD ≥0.5) than the isolates isolated from the invasive infections (OD <0.45). The level of significance for a two-tailed correlation test is 0.05 when OD values of individual isolates of two groups were analysed. Results of this study revealed that biofilm-forming abilities of invasive isolates were less compared to those from non-invasive isolates [Figure 2].
|Figure 2: Biofilm-forming capacity of invasive and non-invasive isolates|
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Twelve isolates out of 37 isolates amplified wchA gene [Figure 3]. Neighbour-joining tree deduced from multiple alignments of wchA sequences by comparing the DNA sequences from eight selected representative isolates resulted in four clusters [Figure 4]. Dendrogram analysis of wchA sequences revealed that cluster-III comprising of three isolates, namely, CMRI-1, CMRI-7 and BMP 241-13 had some interesting and significant findings. It was found that three important point mutations that were common in these three isolates when compared with the referral 554/62 wchA sequence (GenBank accession no. CR931643) at three different positions such as 31, 239 and 319. In position 31, there was a change in amino acid from A to T, at position 239, there was an amino acid change Y to H and finally, at 319 position, the amino acid change was S to I. Alignment of wchA sequences performed through MegAlign showing the point mutations has been illustrated in [Figure 5].
|Figure 3: Screening of wchA in Streptococcus pneumoniae isolates obtained from invasive and non-invasive infections/nasopharyngeal carriage. Lanes: Lane 1-BMP1958, 2-CMRI 1, 3-CMRI 3, 4-CMRI 6, 5-CMRI 7, 6-IBT1960, M-Marker, 7-IBT1975, 8-IBTI793, 9-IBT1721, 10-BJMC 3, 11-BJMC 5, 12-BMP208-13, 13-BJMC 2, 14-BMP241-13|
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|Figure 4: Neighbour-joining tree deduced from comparing the wchA gene sequences from eight selected representative isolates in the study|
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|Figure 5: Multiple alignment of wchA sequences performed by MegAlign showing the single-nucleotide polymorphisms generating non-synonymous mutations leading to change in amino acid|
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| ~ Discussion|| |
Mechanisms of pathogenesis and role of virulence factors in the disease process of S. pneumoniae are poorly understood. The problem is further compounded by the presence of the organism as commensals in healthy individuals. Demonstration of genes coding for some of the virulence factors from isolates causing invasive infection and commensals may help in understanding the mechanisms involved in the disease process. This investigation analysed S. pneumoniae isolates obtained from two different niches, namely, carriage and invasive infections with particular reference to the distribution of virulence genes that can help in understanding the differential capabilities of these two group of isolates in the manifestation of pathogenesis. The study was also interested in looking into whether there is any potential disease-specific virulence factor (s) exists among these two groups.
In the present study, lytA gene that codes for autolysin responsible for cell wall lysis was universally present in both invasive and colonising isolates indicating that autolysin is a necessary absolute requirement for S. pneumoniae. Moreover, N-acetylmuramoyl-L-alanine amidase is the best-characterised autolysin enzymes implicated in the pathogenicity of pneumococci by mediating the cell lysis. On the other hand, ply gene responsible for pneumolysin, an autotoxin was more pronounced among invasive isolates (79%) when compared to non-invasive isolates (52%) predominantly cultured from ocular and nasopharyngeal samples. This was contrary to the findings of an earlier report wherein 90% of isolates obtained from nasopharyngeal samples were positive for the ply gene. Pneumolysin appears to be important for invasive isolates that cause systemic infections than for the isolates those cause superficial ocular infections such as keratitis. Consequent to autolysis induced by LytA, there is the release of cytoplasmic bacterial proteins including virulence factors such as pneumolysin, lipoteichoic and teichoic acids that are the mediators of host inflammatory response. Pneumococcal pneumolysin is a cytolytic protein and its effect contributes to lung injury and neuronal damage while its pro-inflammatory effects compound the tissue damage.
We observed amiA gene to be present significantly higher among the isolates of the non-invasive group. Genes encoding protein-dependent peptide permeases contains five elements that encode a substrate-binding protein (AmiA) and other membrane-associated proteins (AmiC, AmiD, AmiE and AmiF) that function as substrates for transport machinery. An earlier study that used murine pneumonia model reported that S. pneumoniae requires AmiA, for successful colonisation of the nasopharynx. The results of our investigation suggest that the AmiA may not be a significant contributor for the virulence during pneumococcal disease but is likely to play a critical role in colonisation of the nasopharynx. In view of the fact that to cause invasive disease, the pneumococcus must first colonise the nasopharynx that may be followed by subsequent spread to lungs through aspiration.
PsaA is a surface adhesion lipoprotein that helps in adhesion of bacteria onto mucosal cells and another type of host cells. The psaA gene has been demonstrated in all of the 90 serotypes of S. pneumonia. However, in this study, psaA gene did not differ significantly among the two groups investigated. Enolase (eno) represents anchorless surface proteins which promote host proteolytic activity and bacterial migration across the human extracellular matrix. This surface protein found to be present significantly more in invasive isolates (71%) than non-invasive isolates (57%) signifying its need for migration through aiding in crossing the host barriers in invasive infections. Histidine protein kinase ciaH encodes a putative sensor protein that belongs to the family of signal-transducing histidine kinases form a two-component system with CiaR that regulates several pathways, including teichoic acid synthesis, maintenance of cell integrity, resistance to antibiotics, toxins, virulence and host colonisation. As ciaH is vital for multiple activities of pneumococci, it was not surprising to note its presence evenly among the isolates of both the groups.
Capsular polysaccharide is one of the most important virulence factors which forms the basis of more than 93 different serotypes that been documented so far. It is interesting to note only a few of them (8–12) cause invasive disease., Encapsulation helps the pneumococci to invade and escape from the immune response of the host and majority or all the clinical isolates causing invasive diseases are encapsulated as reported in literature., Loss of capsule by either genetic mutation or enzymatic degradation considerably reduces virulence in animal models of infection. Besides S. pneumonia serotype 3 that is highly mucoid strain is known to be most virulent. Point mutations in wchA gene are known to be responsible for the non-typability of invasive S. pneumonia isolates. Three of the S. pneumonia isolates from cluster-III having mutations at the identical position are significant in our study. Interestingly, all these isolates that bear mutations were isolated from the ocular infections. The possibility of variation in capsular nature may contribute to the pathogenesis of ocular infections. The significance of these mutations has to be further analysed on larger sample size across several types of pneumococcal infections to get better insights on these events of point mutations. Hitherto wchA point mutations in non-typable isolates that are associated with NC/conjunctivitis have not been documented. Nevertheless, it is noted that the wchA gene from different serotypes of serogroup 6 (C and D) was distinct from the other serotypes (viz., A and B) of the same serogroup.
Formation of biofilm is required only at some point of time during the process of invasive disease whereas biofilm development in commensal pneumococci is highly critical for adhesion of mucosal epithelial cells. Further, nasopharyngeal commensal isolates may not be competent enough to invade host cells. One study through using genomic microarrays examined the pneumococcal transcriptome wherein they showed that biofilm formation in S. pneumoniae downregulated the genes involved in virulence and biofilm production, and these strains were highly attenuated for invasive disease but not for nasopharyngeal colonisation. Another recent study demonstrated for the first time that pneumococcal adherence (PavB) and virulence factors (PspC) are key players for the interaction of S. pneumoniae with human matricellular glycoprotein thrombospondin-1 (hTSP-1) that mediates adhesion. PavB and PspC are surface-exposed adhesins and virulence factors in pneumococci that exhibit repetitive sequences in their core structure. Moreover, deficiency of PavB and PspC reduces the recruitment of soluble hTSP-1 by pneumococci and decreases hTSP-1-mediated pneumococcal adherence to human epithelial cells. Capsular polysaccharides are likely to shield the function of bacterial short adhesins by sterically preventing the receptor-target recognition, and hence, we speculate any variation in the capsule would serve to expose the surface components of adhesion proteins that could facilitate adhesion.
A previous literature reported that serotype 3 which is highly mucoid on culture plates indicating a thick capsule around bacterium isolated from invasive sources and rarely from other sources., The majority of S. pneumoniae serotypes do not cause invasive disease, but it is likely that biofilm formation in most of them facilitates long-term colonisation of the nasopharynx rather than promoting the development of invasive disease that is supported by our results that demonstrate abundant biofilm production in colonisers but not in invasive isolates. Our results suggest that the capacity to form robust biofilms is not obligatory for virulence, but alternatively, it may contribute to long-term colonisation and spread of the pneumococci. This study gives us some cue in understanding the extent of the contribution of different individual virulence factor for the pathogenesis of S. pneumoniae, and it is also an attempt made to identify potential disease-specific virulence factor (s) among a different group of isolates that may assist us in differentiating a pathogen from a commensal.
| ~ Conclusion|| |
The results of the present study indicate that there could be multiple capsular variations spontaneously arising out due to specific point mutations in genes coding for capsule among the isolates of S. pneumoniae and this may perhaps reveal the inadequacy of capsular typing resulting in increasing number of non- typable isolates reported in recent times.
The authors would like to thank the Department of Biotechnology (DBT), Government of India, for providing infrastructural support under Interdisciplinary Program in Life Sciences Builder Program. JJ is also supported by SRF fellowship by the DBT, Government of India.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]