The uniqueness of the core sialic acid-related gene cluster to the C. sakazakii and some of
C.turicensis genomes hints at a role in the evolution of the virulence of the organism. The predicted
amino acid sequences of the proteins encoded by these nan cluster genes were individually analysed and their phylogenetic relationships observed with closely related Gram-negative bacteria have been indicated in Fig. 4.7 to 4.16. In the case of each of the genes, the C. sakazakii sequences formed an independent cluster of their own, with the other Enterobacteriaceae Cit. koseri,
Franconibacter pulveris and Siccibacter turicensis members clustering on the neighbouring
branches.
In the nanA (Fig.4.7) and nanR (Fig.4.8) phylogenetic trees of predicted amino acid, the C.
sakazakii cluster appeared to evolve on the same branch as C. turicensis. With the others forming a
separate clade Cit. koseri, Franconibacter pulveris and Siccibacter turicensis.
As well as, the nanK (Fig.4.9) and nanT (Fig.4.10) C. sakazakii and C. turicensis clusters appears to have greater phylogenetic distance from the other closely species, with a clear split of the population into two clades, one of them being that of the C. sakazakii and C. turicensis cluster. The nanE gene was found across the Cronobacter genus, and the phylogenetic analysis of the nanE protein sequences (Fig 4.12) revealed the Cronobacter cluster to have a common and closely related evolutionary clade with Cit. koseri, Escherichia coli. K-12 and Edwardsiella trade.
C. sakazakii nanC demonstrated more than 50% homology with Cit.koseri (Figure 4.13) Moreover,
all Cronobacter species were found to have nagA, nagB, and neuC genes as shown in Figures 4.14- 4.16. When these protein sequences were subjected to phylogenetic analysis, it was found that the evolutionary clade of Cronobacter spp. sequences were quite distinct from other closely related members of Enterobacteriaceae which constitute an adjacent clade, through both share the evolutionary lineage. A dissimilar branching pattern has been demonstrated by nan genes sequence from the Cit. koseri when it was subjected to phylogenetic analysis. It implies that there can be distinct evolutionary paths adopted by nan genes in case of the genus Cronobacter.
4.5 DISCUSSION
It has been reported by Joseph et al. (2013) that the region ESA_03609–13 on the genome of C.
sakazakii BAA-894 encodes for the uptake and consumption of exogenous sialic acid. Moreover,
this region was unique to the genome of C. sakazakii based on RAST analysis. This exclusive characteristic is quite intriguing in terms of virulence and epidemiology of Cronobacter species. Sialic acid metabolism might have a role in high prevalence of C. sakazakii infections among infants and neonates. In contrast, the nanE gene have been found located in a distinct site (ESA_00529) separate from the nan cluster site (ESA_03610-12). The same have been observed with other Gram-negative bacteria such as Cit. freundii and Ed. tarda (Vimr 2012). It potentially points towards a distinct evolutionary lineage for the gene. Moreover, genes for NanT inner membrane transporter protein and NanC outer membrane porin are present in all C. sakazakii strains. For that reason, all of them are able to uptake sialic acid from the environment into the cytoplasm of the cell. It is surprising to note that all Cronobacter possess genes for TRAP transporter i.e. siaPQM (Figure 4.4).
These laboratories experiments have confirmed that C. sakazakii is not the only member of
Cronobacter genus which can utilize sialic acid as a source of carbon as some of C. turicensis have
this ability. Colonization by C. sakazakii in the intestinal tract of humans and consumption of sialic acid from the infant formula, breast milk and brain cell might be due to acquisition of genes responsible for utilization of sialic acid (Almagro-Moreno and Boyed 2009).
Growth of C. sakazakii on ganglioside GM1, as shown in Figure 5 b, shows that the bacterium can produce the sialidase enzyme. This finding has not been reported earlier though researchers had carried out gene sequencing in order to detect the nanH gene in the genome of these bacteria. Researchers conducted an extensive research for Asp-box motifs and sialidase RIP as the homology between the genes coding for sialidases is found to be less than 30% (Kim et al. 2011). Growth of C. sakazakii on GM1 also proves that the organism is capable of degrading the ganglioside. Sialic acid residues, glucose, N-acetyl-galactose, and galactose constitute to form GM1. These building blocks are linked through β 1–3 and β 1–4 linkages and are attached to
steroid. Hence, it has been postulated that degradation of GM1 by different lipases (ESA_02127 & ESA_02202), esterases (ESA_00377 & ESA_00776), β-acetyl-hexosaminidases (ESA_02237 and ESA_02655) and β-galactosidases (ESA_01827, ESA_02977 & ESA_03417) results in formation of metabolisable sugar residues thereby enabling the organism to grow on GM1.
An interesting observation made was that nanE and TRAP transporter (siaPQM) were found conserved across the genomes of the Cronobacter genus in contrast with nanA, nanT and nanK (Figure 4.5). GC content of the entire genome of C. sakazakii is 56% which is significantly greater than the GC content of nanC gene i.e. 47.44%. Slight aberration in the GC content values of the
nanE and nanK genes have also been found (63.18%-62.21% respectively) Table 4.2. This is in
agreement with a past observation recorded during an evolutionary investigation of the nan clusters present in members of Enterobacteriaceae such as Yersinia species, E. coli and Salmonella
enterica (Almagro-Moreno and Boyed. 2009). For this reason, it can be stated that nan clusters
might have evolved in these bacteria in a mosaic fashion. These findings point towards the fact that it is highly likely that acquisition or lost of nanC and nanE clusters by the members of
Cronobacter and nanAKT cluster by C. sakazakii could have been the result of the horizontal
transfer events. This research also analysed the nanAKT cluster genes acquisition or loss based on gene location, the high degree of colinearity of the nanAKT cluster alignment between different genomes have been shown in figure 4.5. Furthermore, the whole cluster is located in a certain location flanked by some conserved housekeeping trait (gltB) and starvation gene (sspA) suggesting loss from other Cronobacter spp. instead of separate acquisition events. Moreover, because of close adaptation of intracellular microorganism to the physiologically stable environments of their host cells, a reductive genome evolution happened that led to the loss of some genes not crucial for life within the host. This is called evolution by reduction (Dobrindt and Hacker 2001). Sequence of proteins coded for utilization of sialic acid in C. turensis and C.
sakazakii have also been determined during the phylogenetic analysis (Figures 4.5-4.11). This
finding indicated that the evolution of the nanATKR genes as a lineage in C. sakazakii and certain strains of C. turicensis was independent of closely related Enterobacteriaceae family.
Expression of sialic acid utilisation gene cluster can be affected by levels of nutrients in the
environment since the nanATK gene cluster is found proximate to the stringent starvation gene homologue (sspA, ESA_03615) in C. sakazakii. C. sakazakii also possesses other related genes like
nagA and nagB responsible for the formation of fructose-6-phosphate which is also indicative of
the fact that the organism can utilize sialic acid as a source of carbon or nitrogen. Human milk, brain and GIT serve to be the three main sources of sialic acid in mammals for commensal as well as pathogenic bacteria.
Researchers have found that human milk is a rich source of sialic acid and highest concentrations of sialic acid have been detected in colostrum up to three months after child birth. For that reason, human beings are exposed to sialic acid right from their infancy. It has been proposed that this exposure affects the concentration of sialic acid in brain (Wang et al. 2001). Concentration of sialic acid in cell membranes inside the brain is 20 times higher than its concentration in other mucosal membranes. Especially, the gangliosides of brain contains high concentration of Neu5Ac giving rise to sialyated glycolipids. Similarly, epithelium of human intestine contains high concentrations of sialic acid. Moreover, levels of sialic acid and N-acetylglucosamine that residue in intestinal mucosa of an infant are considerably greater than that of adults (Wang. 2009). There is an intriguing clinical association between the above mentioned sites of sialic acid build up and epidemiology of Cronobacter species. During the course of neonatal meningitis, this bacterium causes NEC and intensive brain damage. High concentration of sialic acid in the above mentioned sites relates with the C. sakazakii infections as majority of neonatal infections with C. sakazakii have been found to occur during infancy. In particular, half of the cases were reported in the first week of birth and three quarter cases were reported within one month (Lai 2001). In addition the sources of sialic acid, PIF products contain sialic acid, but in lesser quantity (<25%) than human milk. In contrast to the form of sialic acid in human milk i.e. oligosaccharide-bound form, infant formula usually contains sialic acid in glycoprotein bound form (Wang et al. 2001).
In summary;
A key finding from the comparative genomic study was the unique cluster of genes in C.
sakazakii and some of C. turicensis encoding for the utilization of exogenous sialic acid.
Since this is also the species most associated with the neonatal meningitic infections, this association could prove to be a crucial link to the pathogenicity of the organism.