Details of the 17 groups of SPI proteins involved in the network.
Abstract
Typhoid infections have become an alarming concern with the increase of multidrug resistant strains of Salmonella serovars. The new pathogenic Gram-negative strains are resistant to most antibiotics such as chloramphenicol, ampicillin, trimethoprim, ciprofloxacin and even co-trimoxazole and their derivatives thereby causing numerous outbreaks in the Indian subcontinent, Southeast Asian and African countries. Conventional and modern methods of typing had been adopted to differentiate outbreak strains. However, identifying the most indispensable proteins from the complete set of proteins of the whole genome of Salmonella sp., comprising the Salmonella pathogenicity islands (SPI) responsible for virulence, has remained an ever challenging task. We have adopted a network-based method to figure out, albeit theoretically, the most significant proteins which might be involved in the resistance to antibiotics of the Salmonella sp. An understanding of the above will provide insight into conditions that are encountered by this pathogen during the course of infection, which will further contribute in identifying new targets for antimicrobial agents.
Keywords
- Salmonella
- Salmonella pathogenicity island
- SicA
- eigen vector centrality
- k-core analysis
1. Introduction
Food-borne infections are quite common and widely distributed worldwide, though there can be several sources of such diseases. Human Salmonellosis or typhoid, causing systemic infection of the human gastrointestinal tract and diarrhoea, is one such common disease caused by
To deal with the threats of multidrug resistance, several health intervention strategies have been undertaken. However, the prospects for finding new antibiotics for several classes of Gram-negative pathogens are especially poor due to the blockades provided by their outer membrane to the entry of some existing antibiotics and expulsion of many of the remainder by their efflux pumps [6]. It has become imperative that the conventional strategies for dealing with such pathogens are less effective or even at times, ineffective completely, to emerge victorious against the strategies for the war waged out by them. In such cases, the complexities posed can be solved by adopting some non-conventional approaches of finding the drug targets for these pathogens. Proteins, being the functional unit of the cell of any living organism, have always been good targets for combating diseases. Diseases, on the other hand, serve as interesting examples of complex protein interactions among several other heterogeneous entities of and between organisms. However, understanding the complexity of such interacting protein partners, especially with respect to the combat against the pathogens, has always been elusive. Thus, analyses of the mosaic mesh or network of interacting proteins, commonly known as protein interaction networks (PINs) can provide sufficient insight to reveal the indispensable virulent proteins for valuable drug targets [7].
Analyses of a PIN, to highlight important and/or indispensable proteins, can be as simple as centrality measurements with respect to the biological scenario. These can start by determining the number of interacting partners of a particular protein to identify its
Again, extracting knowledge of the most indispensable virulence proteins from among the stipulated sets of SPI proteins could be quite insufficient. Thus, we have carried out further analyses of the whole genome of
2. Approach
2.1. Dataset collection
Proteins for 17
The number of proteins from the different genomic islands starting from SPI-1 till -13 and -15 till -18 were 54, 43, 8, 7, 10, 55, 144, 12, 4, 23, 16, 4, 14, 9, 7, 2 and 97, respectively, with all the combined SPI amounting to a total of 502. The total number of protein interactions obtained from STRING v10 were 334, 339, 3, 21, 9, 192, 1193, 12, 6, 69, 19, 1, 19, 5, 3, 1, 343, for the 17 SPI loci mentioned above and 2570 interactions for all of these combined together. The whole genome of
2.2. Interactome construction
All individual protein interaction data, with medium confidence values obtained by default from String 10.0, were imported into Cytoscape version 3.3.0 [15] to integrate and build the interactomes of network comprising SPI-1 till -13 and -15 till -18, individually and all these 17 SPI collectively (AS). The interaction information, weighted by their strength as per STRING, of all the proteins of
2.3. Network analyses
2.3.1. SPI-PIN
All the interactomes of SPI-PIN have been viewed by Cytoscape version 3.3.0 in the form of graphs of aforementioned interconnected proteins. The networks were subsequently analysed via the Cytoscape integrated java plugin CytoNCA [18] to compute values for the network centrality parameters namely EC, DC, CC and BC. Combined scores from different parameters considered in STRING were taken as edge weights for computing CytoNCA scores. Top 20 proteins for each of the centrality measures were taken for drawing Venn diagrams to find common proteins from each measure.
2.3.2. WhoG-PIN
As few (21) nodes out of the whole genome were isolated from the major part of network, these were considered to have less impact on the overall topology and thus ignored. Further analyses were based on the large connected component (LCC) of network comprising 4508 protein partners having 1041182 interactions. The analytical study has been done by using MATLAB version 7.11, a programming language developed by MathWorks [19].
For the primary understanding of the network, the distributions of network degree (k) were plotted by Complementary Cumulative Distribution Function (CCDF). To extract significant information from the topology of the large and complex Whole Genome Protein Interaction Network (WhoG-PIN), knowledge of the role of each protein was derived from the cartographic representation of within-module degree z-score of the protein versus its participation coefficient as per the methodology described by Guimera et al. [20]. Participation of each protein reflected its positioning within own module and with respect to other modules, where modules were calculated based on Rosvall method [21]. To have an idea of the core group of the very specific proteins which might have variety of role to play in the whole genome context, a k-core analysis was performed following the network decomposition (pruning) techniques to produce a sequence of subgraph of gradually increasing cohesion [12].
3. Features of the 17 SPIs
The virulence proteins of
The 59 kb SPI-6 consists of a type VI secretion system (T6SS), the
4. The individual and the combined SPI-PINs
To focus upon the most indispensable proteins of the highly complex virulent phenotype as that of
Amongst the four centrality measures being mentioned above, DC is the most basic as it brings out the involvement of the protein in a large number of interactions in a network. However, in a biological scenario of
SPI | Degree | Betweenness | Closeness | Eigenvector |
---|---|---|---|---|
1 | hilA,iacP,invA,invE,invF, invG,prgH,prgI,prgK,sicA, sipA,sipB,sipC,sipD,spaL, spaO,spaQ,spaR,spaS,sptP, | hilA,invA,invE,invF,invG, prgH,prgI,prgJ,prgK,sicA, sipA,sipB,sipC,sipD,sirC, sitD,spaO,spaR,spaS,sptP, | hilA,invA,invE,invF,invG, prgH,prgI,prgJ,prgK,sicA, sipA,sipB,sipC,sipD,sirC, spaL,spaO,spaR,spaS,sptP, | hilA,iacP,invA,invE,invF, invG,prgH,prgK,sicA,sipA, sipB,sipC,sipD,spaK,spaL, spaO,spaQ,spaR,spaS,sptP, |
2 | ssaG, sscB, sscA,ssaJ,STY1710, STY1709,ssaL,ssaN,ssaQ,ssaU, ssaS,sseC,sseD,yscR,ssaD,ssaM, ssaT,ssaH,ssaV,ssaI, | ssrA,ssaQ,ttrR,ttrC,STY1710, STY1709,ssaG,sscB,sscA,ssaJ, sseC,ssaD,sseD,ssrB,spiA,ssaL, ssaN,ssaU,STY1730,ssaH, | ssaG,sscB,sscA,ssaJ,ssaQ,ssaN, STY1710,STY1709,ssaL,ssaU, ssaS,yscR,sseC,ssaD,sseD,ssaM, ssaT,ssrA,ssaH,ssaV, | ssaG,ssaJ,sscB,ssaL,sscA,ssaN,ssaS,ssaU,STY1710,STY1709,ssaQ,yscR,ssaV,ssaT,sseD, ssaM,ssaH,sseC,ssaD,ssaI, |
3 | fidL,STY4039,slsA,rmbA,mgtC,mgtB, | fidL,STY4039,slsA,rmbA,mgtC,mgtB, | fidL,STY4039,slsA,rmbA,mgtC,mgtB, | fidL,STY4039,slsA,rmbA, mgtC,mgtB, |
4 | STY4452,STY4453,STY4458, STY4459,STY4460,STY4456, STY4457, | STY4452,STY4453,STY4458, STY4459,STY4460,STY4456, STY4457, | STY4452,STY4453,STY4458, STY4459,STY4460,STY4456, STY4457, | STY4452,STY4453,STY4458,STY4459,STY4460,STY4456, STY4457, |
5 | sopB,pipB,pipD,STY1124, STY1125,sigE,sicA,pipA, | sopB,pipB,pipD,STY1124, STY1125,sigE,sicA,pipA, | sopB,pipB,pipD,STY1124, STY1125,sigE,sicA,pipA, | sopB,pipB,pipD,STY1124, STY1125,sigE,sicA,pipA, |
6 | STY0286,STY0287,STY0288, STY0290,STY0291,STY0294, STY0297,STY0302,STY0303, STY0305,STY0313,STY0317, STY0319,STY0320,STY0321, STY0322,STY0323,STY0324, t2582,t2597, | safB,safC,STY0286,STY0294, STY0297,STY0302,STY0313, STY0314,STY0316,STY0317, STY0318,STY0319,STY0320, STY0321,STY0324,STY0352, tcfA,tcfC,tcfD,tinR, | safB,safC,STY0286,STY0287, STY0288,STY0290,STY0291, STY0294,STY0297,STY0302, STY0303,STY0305,STY0317, STY0319,STY0320,STY0321, STY0323,STY0324,t2582,t2597, | STY0286,STY0287,STY0288,STY0290,STY0291,STY0292, STY0294,STY0297,STY0302,STY0303,STY0305,STY0306, STY0307,STY0319,STY0320,STY0321,STY0323,STY0324, t2582,t2597, |
7 | pilL,STY4521,STY4523, STY4526,STY4528,STY4530, STY4534,STY4562,STY4564, STY4569,STY4571,STY4572, STY4573,STY4575,STY4576, STY4577,STY4579,STY4665, STY4666,t4268, | pilL,pilV,STY4521,STY4523, STY4526,STY4530,STY4561, STY4586,STY4592,STY4618, STY4622,STY4644,STY4645, STY4658,STY4664,STY4666, STY4676,STY4678,t4317, tviD, | pilL,STY4521,STY4523, STY4528,STY4530,STY4534, STY4561,STY4562,STY4564, STY4569,STY4571,STY4572, STY4573,STY4575,STY4576, STY4577,STY4586,STY4665, STY4666,t4268, | pilL,STY4521,STY4523, STY4528,STY4558,STY4559,STY4562,STY4563,STY4564, STY4568,STY4569,STY4571,STY4572,STY4573,STY4575, STY4576,STY4577,STY4579,STY4665,t4268, |
8 | STY3281,STY3277,STY3278, STY3279,STY3283,STY3287, STY3289,STY3288,STY3285, STY3290,STY3291, | STY3281,STY3277,STY3278, STY3279,STY3283,STY3287, STY3289,STY3288,STY3285, STY3290,STY3291, | STY3281,STY3277,STY3278, STY3279,STY3283,STY3287, STY3289,STY3288,STY3285, STY3290,STY3291, | STY3281,STY3277,STY3278,STY3279, STY3283,STY3287, STY3289,STY3288,STY3285,STY3290,STY3291, |
9 | t2643,STY2876,STY2877, STY2878, | t2643,STY2876,STY2877, STY2878, | t2643,STY2876,STY2877, STY2878, | t2643,STY2876,STY2877, STY2878, |
10 | STY4826,STY4832,STY4830, STY4822,STY4852,STY4821, STY4849,t4521,STY4834, STY4828,STY4833,STY4829, t2655,STY4851,STY4825, STY4827,STY4823,sefC,sefB, | STY4832,sefC,STY4826, STY4830,STY4843,STY4822, STY4849,STY4852,sefB, STY4821,t4521,STY4834, STY4828,STY4851,STY4833, STY4829,t2655,STY4825, STY4827,STY4823, | STY4832,STY4826,STY4830, STY4822,STY4821,STY4849, STY4834,STY4828,STY4852, t4521,sefC,STY4833,STY4829, t2655,sefB,STY4851,STY4825, STY4827,STY4823,STY4850, | STY4826,STY4830,STY4832,STY4822,STY4852,STY4821, STY4849,STY4834,t4521, STY4828,STY4851,STY4825,STY4827,STY4823,STY4833, STY4829,t2655,STY4850,sefC,sefB, |
11 | cdtB,pagC,envE,STY1879, STY1880,pagD,STY1889, STY1890,STY1891,cspH, msgA,STY1887, | cdtB,pagC,envE,STY1879, STY1880,pagD,STY1889, STY1890,STY1891,cspH, msgA,TY1887, | cdtB,pagC,envE,STY1879, STY1880,pagD,STY1889, STY1890,STY1891,cspH, msgA,STY1887, | cdtB,pagC,envE,STY1879, STY1880,pagD,STY1889, STY1890,STY1891,cspH, msgA,STY1887, |
12 | sspH2,STY2468, | sspH2,STY2468, | sspH2,STY2468, | sspH2,STY2468, |
13 | uxaC,ordL,STY3296,STY3294, STY3295,STY3298,STY3293, uxuA,uxuB,exuT,STY3302, STY3303, | uxaC,ordL,STY3296,STY3294, STY3295,STY3298,STY3293, uxuA,uxuB,exuT,STY3302, STY3303, | uxaC,ordL,STY3296,STY3294, STY3295,STY3298,STY3293, uxuA,uxuB,exuT,STY3302, STY3303, | uxaC,ordL,STY3296, STY3294,STY3295, STY3298,STY3293,uxuA, uxuB,exuT,STY3302, STY3303, |
15 | STY0605,gtrB,gtrA,STY3188, STY3189,STY3192,STY3193, | STY0605,gtrB,gtrA,STY3188, STY3189,STY3192,STY3193, | STY0605,gtrB,gtrA,STY3188, STY3189,STY3192,STY3193, | STY0605,gtrB,gtrA,STY3188,STY3189,STY3192,STY3193, |
16 | STY0605, gtrB, gtrA | STY0605, gtrB, gtrA | STY0605, gtrB, gtrA | STY0605, gtrB, gtrA |
17 | gtrA2,STY2629, | gtrA2,STY2629, | gtrA2,STY2629, | gtrA2,STY2629, |
18 | barA,cpxR,csrA,flag,flhA,flhB, fliA,fliF,fliH,fliJ,fliP,fliQ,fliR, fliZ,phoQ,rcsB,rcsC,rpoS, STY1297,yojN, | acrR,baeR,barA,clpP,csrA,dnaK, flag,fliA,hns,mgtA,mntH, ompF,phoQ,rcsB,rcsC,rpoN,rpoS,soxS,STY1297,STY1678, | barA,clpP,cpxR,csrA,dnaK,flag, fliA,groL,hns,ompR,phoB,phoQ, rcsB,rcsC,rpoN,rpoS,sirA, STY1297,STY1678,yojN, | barA,flag,flhA,flhB,flhD, fliA,fliF,fliH,fliI,fliJ,fliO, fliP,fliQ,fliR,fliZ,rcsB, rcsC,rpoS,STY1297,yojN, |
All SPI | pilL,STY4521,STY4523, STY4526,STY4528,STY4530, STY4534,STY4562,STY4564, STY4569,STY4571,STY4572, STY4573,STY4575,STY4576, STY4577,STY4579,STY4665, STY4666,t4268, | barA,pilL,pilV,rpoS,sicA, STY4521,STY4523,STY4526, STY4561,STY4586,STY4592, STY4618,STY4622,STY4644, STY4645,STY4658,STY4664, STY4666,t4317,tviD, | pilL,STY4521,STY4523, STY4528,STY4530,STY4534, STY4561,STY4562,STY4564, STY4569,STY4571,STY4572, STY4573,STY4575,STY4576, STY4577,STY4586,STY4665, STY4666,t4268, | pilL,STY4521,STY4523, STY4528,STY4558,STY4559,STY4562,STY4563,STY4564, STY4568,STY4569,STY4571,STY4572,STY4573,STY4575, STY4576,STY4577,STY4579,STY4665,t4268, |
There have been three clear trends observed across the topmost rankers of the SPI-PINs for the measures of DC, BC, CC and EC, respectively. In most of the cases, there is a unanimous decision for the top ranking protein showing its utmost importance nearing to indispensability. SPI-PINs of these categories are -1, -3, -4, -5, -7, -8, -9, -10 to -13 and -15 to -17. The other categories have either three or two of the centrality measures conforming to the unanimosity of the top ranking proteins. SPI-2, -18 and the all SPI (AS-PIN) have BC differing in the top ranking position whereas SPI-6 and -10 have segregation of DC and EC against CC and BC for the top ranking positions. The common top ranking proteins across these 17 SPI and the AS has been reflected in Figure 1 with Venn diagrams.
It has been observed that with SPI-1, protein HilA is ranked highest. HilA is the central regulator in SPI-1, which activates the sip operon that is responsible in encoding secreted proteins, as well as the
With respect to the above analyses of the individual interactomes of the SPI, an idea about the importance of these proteins in their individual SPI and finally across all SPI could be obtained. However, for a drug to be effective, the indispensability issue of these proteins needs to be taken care of. Thus, a broader picture with respect to the whole genome proteins of
5. Feature of the WhoG-PIN
It is imperative that the WhoG-PIN, built from the empirical and theoretical results of physical and functional interactions among proteins laid down in STRING, can be random like that proposed by Erdos and Renyi [53] or a small-world type proposed by Watts and Strogatz [54]. The idea was to see if the connectivity distribution,
6. Decomposition of WhoG-PIN
In order to get an idea of the indispensable ones from the barrage of proteins involved in the individual SPI-PINs and AS, we have performed a k-core analysis for them. A k-core is a subgraph whose nodes have degree at least equal to k. Nodes which are part of k-core, but not in the k+1 core, is called, k-shell. This is able to classify the nodes (proteins, in our study) based on the variety of their interacting partners. Proteins, which belong to outer shell, have lower k value and thus reflect limited number of interacting partner proteins. Moreover, proteins, which belong to inner k-core/shell, are specific ones, highly interacting with each other and thus can be considered to be the most important ones. Decomposition of this core decomposes the network and thus makes this the innermost core.
After decomposition of the WhoG-PIN, we have obtained the inner core member proteins which are highly robust, central and thus highly interactive in nature [56]. We have arrived to the 154th core with a number of 2180 proteins (Figure 3; data not shown). An idea was to look in for the rank holder proteins of the AS-PIN obtained through the EC, DC, CC or BC measures. Interestingly, it was found that the top ranker PilL, across EC, DC and CC measures, belong to the 111th core and not the 154th core. On the contrary, the top ranking BC protein, BarA, was in the 154th core along with the closely ranked PilV in the 150th core. The only other protein, amongst the unanimous top rankers of AS-PIN, STY4521 had a position of 145 in k-core measures. Very strikingly, two proteins of BC top rankers were also in the 154th innermost core along with BarA. These were the RNA polymerase sigma factor, RpoS and the chaperone protein, SicA. On a note of comparison among the top ranking proteins of EC and BC analysed for AS-PIN, proteins of the latter group had higher ranks in the whole genome context, with STY4586, STY4644 and STY4664 having the same 154th innermost core measures. On the contrary, those from the former ranking group (EC) mostly moved around the core numbers 56–70. This reflected that proteins from the BC rankers were more important in their interaction with other proteins, forming a bridge amongst those and thereby rendering high betweenness.
In an earlier work by Lahiri et al., SicA was found to be in the group of innermost core of the interactome comprising the five most extensively worked out SPI of
7. Cartographic analyses of WhoG-PIN
For the purpose of classification of the proteins of
As can be understood from the name itself, a hub is a connection point of many nodes. The category of non-hub nodes can be assigned four different roles namely, R1 comprising ultra-peripheral nodes, R2 of peripheral nodes, R3 of non-hub connector nodes and R4 having the non-hub kinless nodes. Likewise, the hub nodes can be assigned three different roles namely, R5 of provincial hubs, R6 of connector hubs and R7 of kinless hubs (Figure 4). The kinless hubs nodes are supposed to be important in terms of functionality, which has high connection within module as well as between modules. Accordingly, the ultra-peripheral nodes occupy the least connecting position in the network followed by the peripheral nodes. These nodes can be pruned easily without much affecting the whole network while decomposing it to reach the core (refer previous section for k-core). The non-hub connectors are expected to take part in only a small but fundamental set of interactions. This is just opposite to those of the provincial hubs class which have many within-module connections. The non-hub kinless nodes are those with links homogeneously distributed among all modules. The most conserved in terms of decomposition as well as evolution would be, however, those from the connector hubs with many links to most of the other modules. The system would try to retain these connections as essential ones for their very survival.
As can be perceived from the above classification of the connectors and the hubs, the proteins belonging to the R4, R6 and R7 role players are very crucial and can be regarded as potential drug targets. In the context of our WhoG-PIN, the only one R7 is a putative transposase, STY0115 and reminds of the Tn5 transposase, the enzyme that helps bacteria to share antibiotic resistance genes [58, 59]. This is closely followed by the plasmid transfer protein, TrhC in R6 group. This could very well play a good target for drugs as plasmids are known to be powerhouse of the antibiotic resistance genes [60]. Uncoupling of phosphotransferase system could also be an effective way of getting targets for novel drugs as exemplified by PtsG, TreB, NagE and t0287 [61]. Inhibition of glutamate Synthase, GltB has already been utilized as target for
Protein name | R | Description of function |
---|---|---|
STY0115 | 7 | Putative transposase |
trhC | 6 | Plasmid transfer protein |
gltB | 6 | Glutamate synthase (NADPH) large subunit |
ptsG | 6 | PTS system glucose-specific transporter subunit IIBC |
hemE | 6 | Uroporphyrinogen decarboxylase |
nagE | 6 | PTS system N-acetylglucosamine-specific transporter subunit IIABC |
STY3507 | 6 | Aerobic respiration control sensor protein |
t0287 | 6 | PTS system sucrose-specific transporter subunit IIBC |
treB | 6 | PTS system trehalose-specific transporter subunit IIBC |
Cat | 4 | Chloramphenicol acetyltransferase |
pspF | 4 | Phage shock protein operon transcriptional activator |
STY4151 | 4 | Acetyltransferase |
STY4518 | 4 | Acetyltransferase |
STY4668 | 4 | Hypothetical protein with Acetyltransf domain |
STY4678 | 4 | Integrase |
STY4680 | 4 | Integrase |
STY0326 | 4 | Hypothetical protein |
STY3695 | 4 | DNA-invertase |
modB | 4 | Molybdenum transporter permease |
STY4017 | 4 | Putative transferase |
modA | 4 | Periplasmic molybdenum-binding protein |
sopE | 4 | Guanine nucleotide exchange factors |
STY1020 | 4 | Sequence-specific DNA binding |
STY3193 | 4 | Hypothetical protein |
ugpB | 4 | Glycerol-3-phosphate-binding periplasmic protein |
tviA | 4 | Flagellar regulator |
hpaG | 4 | Isomerase/decarboxylase |
STY4175 | 4 | Hypothetical protein |
ratA | 4 | CS54 island protein |
livG | 4 | High-affinity branched-chain amino acid transport ATP-binding protein LivG |
STY0352 | 4 | Periplasmic protein |
8. Conclusion
This work schematically delineates a process of figuring out the most indispensable protein in a system of interacting proteins of
Acknowledgments
The authors wish to acknowledge the support of IMSc, Chennai and Dept. of Computer Applications at BSAU, Chennai for the provision of computational facilities. The personal contribution of Ong Su Yean for the SPI data and of Indrajeet Chakraborty for the formatting are highly appreciated and acknowledged.
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