Background
Salmonellosis is one of the leading causes of mortality among infectious diseases [
1].
Salmonella enterica are Gram-negative facultative intracellular pathogens bearing around 2400 serovars which either have certain host specificity or infect a huge variety of hosts [
2]. Annually nearly a million deaths are attributed to enteric pathogens; henceforth the health concerns rise for the development of targets for drug design [
3].
Salmonella consists of two species namely
S. bongori and
S. enterica. The two epidemiologically significant serovars are
Salmonella enterica serovar Typhimurium (
S. Typhimurium) and Enteritidis (
S. Enteritidis) [
4]. Statistics show the prevalence of
S. Enteritidis infections to be 43% as compared to 19% of
S. Typhimurium. These infections have raised the bar of concern for public health. Hence this has led to the identification of multiple virulence factors and pathogenicity islands for determining their role in virulence [
5].
S. enterica infects host cells with its supramolecular needle-like complex called the Type Three Secretion System (T3SS) [
6]. This is encoded by
Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) for invasion into epithelial cells and survival within phagocytic cells respectively. Both SPI-1 [
7] and SPI-2 [
8,
9] are essential factors for
Salmonella pathogenesis which is mediated through the secretion of effectors via their respective T3SS. The injection of effectors into host cells leads to actin cytoskeletal rearrangements and membrane ruffling, thereby internalizing the bacterium and further mediating pathogen survival and infection persistence [
10,
11]. Though SPI-1 and SPI-2 still remain as crucial determinants of virulence; other SPIs and non-SPI genes have also shown significance in
Salmonella pathogenesis. The deletion of such virulence determinants would aid in analyzing their host interaction or contribution to microbial adaptation [
12].
Salmonella enterica serovar Enteritidis (
S. Enteritidis) and Typhimurium (
S. Typhimurium) cause non-typhoidal self-limiting gastroenteritis with symptoms of fever and diarrhoea in humans [
13]. A microarray analysis identified genes essential for colonization and pathogenesis of
S. Typhimurium both in vitro and in vivo models [
14‐
17]. Another study reported the fast invasion kinetics and SPI-1 dependent inflammation of
S. Enteritidis in streptomycin pretreated C57BL/6 murine models as compared to SPI-1 deficient
S. Typhimurium strain [
18,
19]. Hence the identification of genes responsible for early colonization of
S. Enteritidis would be of significant relevance. A study reported that there are certain additional genes present in
S. Enteritidis for infection establishment as compared to
S. Typhimurium [
20]. This led to the hypothesis of the involvement of these genes in
S. Enteritidis contributing to greater colonization and host–pathogen interaction. A previous study from our laboratory involving a comparative genome analysis of Enteritidis and Typhimurium serovars showed 98% identity with 2% extra genetic elements likely being involved in the greater infection phenotype of
S. Enteritidis [
18]. Both serovars share similar virulence mechanisms pertaining to epithelial cell invasion and survival within macrophages. In spite of these similarities, both exhibited variations in their infection profiles relating to pathogen persistence in host. It was found that more than 200 differential genes were present in
S. Enteritidis as compared to Typhimurium most of which were clustered in unique islands gained through horizontal gene transfer termed as “regions of difference” (ROD).
The objective of the present study is to investigate the role of SEN3897, an alanine racemase gene that showed significant differential expression in ROD34 island of
S. Enteritidis which wasn’t present in
S. Typhimurium. Alanine racemases (EC 5.1.1.1) are unique pyridoxal-phosphate dependent bacterial enzymes essential for the reversible racemization of
l-alanine to
d-form [
21,
22]. They are ubiquitously present in all prokaryotes and also exceptionally present in eukaryotes like fungi and yeasts to produce
d-alanine for peptidoglycan synthesis. Generally either the presence of one or two alanine racemase genes have been reported [
23‐
25]. For example in
Salmonella Typhimurium; there are two isoforms (non-homologous) of alanine racemase genes (
alr and
dadX). The constitutively expressing alanine racemase gene;
alr is essential for cell wall synthesis by forming
d-alanine required for peptidoglycan cross-linking [
26]. Another gene
dadX forms a secondary source for cell-wall biosynthesis though it’s basically required for
l-alanine catabolism forming a substrate for
d-alanine dehydrogenase (
dadA). Nonetheless in
S. Enteritidis; in addition to these two conserved alanine racemase genes, the presence of an additional alanine racemase gene (SEN_
3897) was quite interesting and speculative in terms of its functional relevance in the pathogen. Moreover bacterial cell wall has always been an interesting target for many antibiotics and antimicrobial agents [
27]. Hence this study targets to characterize this uniquely differential alanine racemase gene in
S. Enteritidis and investigate its role in virulence with a view that it may serve as a potential therapeutic target.
Discussion
Due to the development of drug-resistant enteric pathogens, novel drug design requires improved formulations and strategies. Bacterial cell walls have always been a fascinating target for the development of new antimicrobials [
44]. Alanine racemase is an essential prokaryotic homodimeric PLP dependent enzyme catalyzing the reversible racemization of
l- to
d-alanine.
d-alanine forms an integral component for peptidoglycan synthesis and is therefore essential for bacterial growth and survival.
Salmonella Typhimurium comprises of two isoforms of alanine racemase genes namely
alr and
dadX [
26]. The constitutively expressing Alr has the primary biosynthetic function of cell wall synthesis and DadX acts as the catabolic alanine racemase which can secondarily act for peptidoglycan cross-linking [
45,
46]. Genomic comparison between
S. Enteritidis and
S. Typhimurium led to the identification of a third alanine racemase in
S. Enteritidis. The presence of a third racemase gene was quite intriguing as the presence of either one or two genes has been reported in every microbe [
23,
24]. This study has discussed the characterization and contribution of three alanine racemase genes of
Salmonella Enteritidis with a focus on this newly identified racemase gene SEN3897. This is the first study to report the presence of a third alanine racemase gene in
Salmonella Enteritidis. The functional role of this unique alanine racemase gene (SEN
3897) in
S. Enteritidis was studied through its sole chromosomal inactivation and presence irrespective of other known alanine racemases to investigate its distinguished role in the pathogen.
Among the mutant strains (SEN
∆alr, SEN
∆dadX, SEN
∆3897), the double mutant
SEN∆
alr∆
dadX (sole presence of SEN3897) displayed signs of growth defects, membrane integrity loss and structural modifications with the depletion of
d-alanine pools. This indicated that the presence of SEN3897 only couldn’t entirely contribute to
d-alanine prototrophy. One similar study had also investigated cellular damage and enhanced permeability due to
d-alanine auxotrophy in
A. hydrophila alanine racemase mutant [
47]. Depletion of intracellular
d-alanine amounts had similarly resulted in cell death with decreased optical density in many Gram-positive and Gram-negative bacteria like
E. coli, Bacillus subtilis and
Lactobacillus plantarum [
22,
41,
43,
48‐
51]. DadX forms a secondary source for
d-alanine production in the absence of Alr. Nevertheless SEN_3897 seemingly fails in
d-alanine production in the absence of both Alr and DadX. This shows that the
alr gene when inactivated probably requires either another copy/isoform for survival or
d-alanine supplementation in the growth medium. Some studies have reported a single deletion of
alr gene to be responsible for
d-alanine auxotrophy in
Lactobacillus plantarum or a double knockout of two essential alanine racemases to show
d-alanine dependency in
Salmonella Typhimurium [
41,
52]. However our study showed the contribution of a unique alanine racemase gene in strict
d-alanine dependency which differs from other related findings.
Alanine racemase genes have been well identified, cloned and expressed in varied microorganisms [
24,
26,
45]. Our results presented that the specific activity of SEN3897 was higher than Alr and DadX. The results also indicated SEN3897 to be less reactive with
l-alanine relative to
d-alanine. Alanine racemases undergo reaction catalysis with a “two-base” mechanism by utilizing two active site residues, i.e., lysine and tyrosine for substrate recognition and conversion [
53‐
55]. The active site of Alr in
S. Enteritidis is formed by two catalytic residues Lys34 and Tyr343 from two monomers [
45,
56] which are conserved among different Alrs in microbes [
57,
58]. Similarly SEN3897 contains Lys42 and Tyr349 catalytic active site residues whereas DadX contains only Tyr 449 (Additional file
5: Table S2).
Alanine racemases are ubiquitously present in all prokaryotes and their deletion has been reported to be lethal for pathogen survival in the absence of
d-alanine supplementation [
22,
41]. Impairment of intracellular survival in macrophages has also been witnessed in alanine racemase mutants or during nutrient deprivation in phagocytes [
42]. Our in vitro survival assays in macrophages displayed the role of biosynthetic alanine racemase gene (
alr) in survival rather than the catabolic gene (
dadX).
d-Alanine catabolism for ATP generation could possibly be carried out by other alternative metabolic pathways in the DadX mutant. However Alr is majorly involved in cell wall biosynthesis; therefore deletion of Alr was expected to cause disruption in cell wall formation thereby affecting growth and viability. Alr mutants of
M. smegmatis have
d-alanine dependency [
49]. Additionally these mutants of
M. smegmatis have 80% reduced survival in macrophages [
43]. The inactivation of essential metabolic genes in SEN
∆alr∆dadX led to diminished intracellular survival of pathogens in our study. This is mostly due to nutrient limitation in the phagosomal compartment as compared to the rich cytosol [
59‐
62] which couldn’t be restored even with the presence of SEN
3897 in SEN
∆alr∆dadX. Furthermore
d-alanine deficiency affected
Salmonella’s entry into the rich host cytosolic compartment in SEN
∆alr∆dadX that was subsequently restored with
d-Ala addition to cell culture media. Hence, this study showed that the sole presence of SEN3897 was responsible for
d-alanine auxotrophy, morphological alterations and reduced pathogen survival. Further a double mutant of
alr and SEN
3897 was also investigated for the additive effects of mutation in contributing to invasion and survival within epithelial cells and macrophages respectively in vitro. Interestingly, SEN
∆alr∆3897 showed comparable phenotypes with WT in terms of epithelial cell invasion (Additional file
11: Figure S9A) and macrophage uptake at 2 h post infection (Additional file
11: Figure S9B). However this double mutant displayed approximately 1.5-fold increased survival within macrophages at 24 h p.i. relative to WT (Additional file
11: Figure S9C). Since both SEN
∆alr and SEN
∆3897 single mutants showed attenuation within epithelial cells and macrophages, a double mutant of these two genes may be expected to show an attenuated profile within the same cell lines in vitro. However the variation in the phenotypes between the individual mutants and their double mutant possibly suggests the involvement of complex multi-pathway interactions that displayed synergistic effect of mutations affecting the bacterial system for adapting to its host environment. Additionally the presence or absence of
dadX by itself didn’t affect colonization levels in SEN
∆alr∆3897 and SEN
∆dadX respectively indicating that DadX (catabolic alanine racemase) may not be crucial for
Salmonella pathogenesis. Comparatively the presence of either
alr or SEN
3897 with
dadX in SEN
∆3897 and SEN
∆alr respectively showed reduced colitis which indicates the essential involvement of SEN
alr and SEN
3897 in virulence.
In
Salmonella, SPIs are known to play a pivotal role in epithelial cell invasion and intracellular survival [
7,
63,
64]. Hence we investigated if the mutants of
alr and SEN
3897 could possibly affect SPI-1 and SPI-2 through this regulatory pathway for its invasion-defective and attenuated survival phenotype respectively. H-NS is a global repressor of SPI-1 regulators (HilA, HilD, HilC, RtsA) [
65] as well as SPI-2 machinery (
ssaB,
ssaG). H-NS silences
hilA expression which affects the invasion genes [
66]. HilA plays an essential role in invasion since its inactivation affects the entire SPI-1 locus [
67,
68]. This transcriptional activator induces InvF expression and together they coordinate regulated control over SPI-1 effectors [
69]. The invasion genes are also regulated by HilD which activates HilA in vitro [
70]. We observed a down-regulation of these essential SPI-1 regulators and effectors in SEN
∆alr and SEN
∆3897. Similar to SPI-1, we observed significant down-regulation of crucial SPI-2 effectors (
sseJ,
sseG) in SEN
∆alr and SEN
∆3897. Effectors like SseJ and SseG contribute to successful
Salmonella replication by inducing SIF formation and stabilizing SCV membrane integrity [
66,
71]. SseJ is identical to acyltransferase/lipase which localizes during infection to affect SIF formation [
72,
73]. SseF and SseG share sequence similarity and are associated with SCV migration in proximity to Golgi apparatus [
74]. Hence our work suggests a combined effect of nutrient deprivation and SPI modulation by SEN3897 to affect pathogen survival in vitro. We also found increased expression of
hns contributing to reduced levels of
hilA and other downstream effectors in
SEN∆
alr∆
dadX which explained the reduced phenotype of epithelial cell invasion in the
alr dadX double mutant. Additionally, in this double mutant, the SPI-2 genes (
sseJ,
sseG) also showed immense down-regulation compared to the transcript levels of these crucial effectors observed in
SEN∆
alr and SEN∆
3897.
Our in vivo studies portrayed attenuation in gut inflammation of SEN
∆3897 at 72 h p.i. relative to WT. The attenuation in intestinal inflammation could be possibly due to the reduced replication and survival of SEN
∆3897 in murine macrophages. However there wasn’t any sort of attenuation observed in cecal colonization possibly due to the differences in host system. It has been reported that SPI-1 is likely to be responsible for attenuation in gut inflammation and inflammatory responses via the needle complex, T3SS-1 [
75]. This study showed decreased expression of the crucial SPI-1 effectors in SEN
∆3897 relative to WT which likely resulted in reduced inflammatory responses in vivo. SPI-2 is also essential modulator of host inflammatory responses [
75]. SEN
∆3897 also showed a down-regulation in the expression of SPI-2 effectors (
sseJ,
sseG). Hence investigation into its coordinated regulation by different pathogenicity islands and regulators will help us develop novel insights that may be exploited for development of antibacterial therapy against
Salmonella. This study portrays a complex network of regulators and virulence-associated factors governing pathogen’s invasion and survival strategies within host [
66]. In vivo, the
alr dadX double mutant displayed hyperattenuation in which the
d-alanine synthesis route was possibly inactivated that led to its defective survival and colonization at 72 h p.i. Moreover if
d-Ala was present in murine macrophages and mice models; their levels are much below threshold to support growth of the
d-alanine auxotrophic strain which lacked the essential alanine racemase genes
alr and
dadX for infection establishment. Hence the major finding of this study stated that the sole presence of the third alanine racemase gene; SEN
3897 couldn’t support pathogen survival in vivo and led to strict
d-alanine auxotrophy. Moreover deletion of this gene led to defective SPI-1 and SPI-2 functioning; hence this work helped in understanding the link between metabolic genes and their role in
Salmonella virulence. Cell wall auxotrophy was also studied for reduced pathogenesis in
Shigella [
76].
The first drug for alanine racemase was cycloserine [
24,
77]. Even though Alanine racemase is a key target for antibacterial drug design, the inhibitors of alanine racemase do not have clinical utility because of their lack of specific targets which promotes activity against other PLP-dependent proteins. The Alr inhibitor DCS (
d-cycloserine) had major health implications on human nervous system which necessitated the requirement for novel inhibitors with better specificity and less host toxicity. Subsequently the inhibitor development through structure-based studies would examine the dimer interfaces and active site pockets of alanine racemases for use as potential targets against microbial infections. The inhibitor-protein interaction should be aimed for the discovery of new antibiotics with high selectively and specificity for alanine racemase. Another study examined a
d-alanine auxotroph as a live vaccine candidate against
Staphylococcus aureus infection [
78]. Our study also examined the role of a
d-alanine auxotrophic strain in
Salmonella pathogenesis. The growth of the
d-alanine auxotroph is dependent on the availability of crucial metabolites in the host tissues. The double mutant auxotroph of
S. aureus hence showed increased pathogenic loads in host cytosol due to
d-alanine supplementation during the course of infection [
78]. However, removal of
d-alanine restored the attenuated profile of the auxotroph which was also observed in our study. Similar model could be replicated in vivo for modulating the virulence phenotype of the
d-alanine auxotroph causing transient pathogenic survival for induction of strong immune responses. An auxotroph of
Listeria monocytogenes was observed to lack immunogenic potential without
d-alanine supplementation hence the vaccination strategy required the addition of the bacteria together with
d-alanine [
48]. Hence an urgency for vaccine production is vital by utilizing modified immunization strategy against these microbial threats.