The continuous battle for survival in the environment has led to the development or acquisition of sophisticated defence systems in bacteria. These defence systems have contributed to the survival of the bacterial species in the environment for millions of years. Some systems appear to have evolved in a number of pathogenic bacteria towards a role in virulence and host immune evasion. Recently, different bacterial cell envelope components from diverse bacterial species have been linked not only to bacteriophage defence, but also to virulence features. In the present review we focus specifically on the bacterial cell envelope-expressed sialic-acid-containing LOS (lipo-oligosaccharide) structures and Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) genes that both occur in specific Gram-negative pathogens. In Campylobacter jejuni circumstantial evidence points at a potential intertwined dual function between sialylated LOS structures and subtype II-C CRISPR–Cas, i.e. in phage defence and virulence. In the present review we discuss whether a dual functionality of sialylated LOS and subtype II-C CRISPR–Cas is exclusive to C. jejuni only or could be more widespread within the group of Type II CRISPR–Cas-harbouring bacteria. We conclude from the literature that, at least in C. jejuni, circumstantial evidence exists for a complex intertwined dual functionality between sialylated LOS and Type II CRISPR–Cas, and that other bacteria show similar genomic signatures.

Introduction

In the environment bacteria are continuously exposed to biotic and abiotic stressors. Bacteria can fall prey to lytic bacteriophages or can be engulfed and destroyed by amoebae [1]. Bacteria also have to deal with diverse abiotic stress stimuli, including sudden changes in temperature, e.g. during infection of host, exposure to antibiotics, UV light and drought [1]. Bacteria have evolved or acquired usually tightly regulated stress-resistance mechanisms that are important for survival in their environmental niche [1]. Some of these acquired or evolved mechanisms may provide an additional advantage when bacteria infect vertebrates [1]. For example, bacterial survival mechanisms activated upon engulfment by amoebae may also provide efficient protection to the same bacterial pathogens upon invasion of eukaryotic cells or upon phagocytosis by macrophages [27].

The bacterial cell envelope is one of the key structures involved in counteracting and responding to biotic [8] and environmental [9] stresses. Interestingly, results from independent studies focussing on Azotobacter spp., Bordetella spp., Escherichia coli, Pseudomonas spp., Salmonella spp. and Staphylococcus aureus appear to suggest that modification of the bacterial cell envelope as a defence mechanism against lytic bacteriophages also contribute to the virulence of these bacteria. For example Protein A, a cell-wall-anchored virulence factor of S. aureus, interacts with the Fc region of human IgG. Absence of Protein A was found to enhance bacteriophage binding to S. aureus, a strong indication that Protein A blocks a bacteriophage-binding receptor [10,11]. The lipoprotein OmpA of E. coli is an important receptor for many T-even-like coliphages, but it is also an important virulence factor [12,13]. When OmpA-bearing E. coli strains harbour a plasmid that encodes the outer membrane protein TraT, OmpA is blocked or modified, protecting E. coli against infection by the T-even-like coliphages [14,15]. Pseudomonas and Azotobacter species express EPSs (exopolysaccharides) that provide protection from the surrounding environment. EPSs block access to receptors that can be bound by some bacteriophages which would then lead to infection of bacteria. During this ‘arms race’, phages have emerged that use these sugars during their infection process, or express enzymes able to dissolve the EPSs, making receptors available again for binding, enabling bacterial infection [1620]. Also Salmonella and E. coli species use complex EPS structures on the bacterial cell envelope to avoid binding by bacteriophages during phase variation, and the same EPS decorations can be exploited by some bacteriophages for cellular entry [2123].

Specific environmental, envelope stress and bacterial membrane physiology have been indirectly linked to a described bacteriophage defence system, CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) [2428]. In 2002, it was observed that in bacteria and archaea, a repetitive element called CRISPR was usually accompanied by a set of genes [29]. A few years later it was proposed by three independent groups that the CRISPR–Cas system might be involved in bacteriophage defence, on the basis of observations that some of the repetitive DNA sequences in the CRISPR array displayed 100% sequence identity with bacteriophage protospacers [3032]. In 2007, it was experimentally established that the Type II CRISPR–Cas system in Streptococcus thermophilus functioned as a bacteriophage defence system [33]. Hereafter, it became firmly established that the CRISPR–Cas system functioned in phylogenetically extremely diverse bacteria as a defence system against foreign DNA [24,3436]. CRISPR–Cas provides defence against bacteriophages by activating a double-stranded nucleic acid degradation response upon detection of bacteriophage RNA or DNA. Recognition of phage DNA is based on identity between bacteriophage sequences and short (approximately 30 bp) DNA fragments represented by CRISPR spacers located in the bacterial genome. Clear examples of the roles CRISPR–Cas may play during a stress response have been established in the bacterial species Myxococcus xanthus and E. coli [37,38]. In M. xanthus, environmental stress was concomitant with the activation of the CRISPR–Cas system and bacterial sporulation [37]. In E. coli CRISPR–Cas was activated when E. coli bacteria were subjected to cell envelope stresses [38]; downstream activation was under the control of transcriptional stress regulators [39].

The CRISPR–Cas bacteriophage defence systems are divided into three main groups, which are based on the presence of group-specific genes [28]. The Type II CRISPR–Cas system, exemplified by the system originally observed in Neisseria meningitidis, is strongly reduced in size, compared with Type I and Type II CRISPR–Cas systems. Type II CRISPR–Cas contains two type-specific cas genes, csn2 or cas4, in addition to the conserved cas genes cas9-1-2 [28,40] that allow a further division into three subtypes [41]. Interestingly, the Type II CRISPR–Cas system seems to be overrepresented in pathogenic bacteria that are able to incorporate sialic acid into their cell envelope [40]. In the present review we discuss a potential dual role in viral defence and virulence for two bacteriophage defence systems that are together present in specific Gram-negative pathogens: cell envelope sialylation and Type II CRISPR–Cas.

Sialic-acid-mediated viral defence

The cellular membranes of vertebrates and, to a lesser extent, bacteria, fungi and plants, incorporate sugar structures that harbour the molecule N-acetylneuraminic acid, also known as sialic acid [42]. In humans, sialylated sugar structures are determinants of susceptibility to viruses, e.g. mumps virus [43] and influenza virus [44,45]. The interactions between influenza viruses and mammalian sialylated sugar structures have been well studied, and the insights from these studies have led to the development of drugs that are able to inhibit influenza release from infected cells by blocking the viral neuraminidase, an enzyme that cleaves terminal sialic acids enabling the discharge of new influenza viruses [4548]. Some Gram-negative pathogenic bacteria, including C. jejuni, also decorate their LOS (lipo-oligosaccharide) structures with sialic acid residues, with relevance to pathogenicity [49,50].

C. jejuni, a zoonotic human pathogen, is the leading cause of diarrhoea worldwide, with 400–500 million laboratory-confirmed cases each year [51]. C. jejuni strains can be divided into two separate groups on the basis of the presence or absence of sialic acid on their outer surface LOS carbohydrate residues [52,53] (Figure 1). Transfer of sialic acids is mediated by the sialyltransferase Cst-II [5456]. Sialylated C. jejuni isolates are highly pathogenic [49,50], cause severe colitis [57] and are associated with reactive arthritis and paralytic diseases such as GBS (Guillain–Barré syndrome) [57,58], a post-infectious life-threatening complication. GBS is suggested to be provoked by molecular mimicry between the sialylated carbohydrate LOS structures of C. jejuni and ganglioside epitopes expressed on human peripheral nerves, resulting in autoantibody-induced and complement-mediated nerve destruction [52]. A recent study using 19 different C. jejuni isolates and two (complemented) mutants competent or disabled in the ability to express ganglioside-like sialylated LOS structures revealed that the C. jejuni ganglioside-like LOS structures provide global protection against most of the lytic bacteriophages that use C. jejuni as host [59]. In this study, it was shown that sialic acid did not function as a phage receptor, but rather provided a defence mechanism that protected C. jejuni from bacteriophage infection [59], most probably by blocking bacteriophage-produced polysaccharide-degrading enzymes or by blocking compatible bacterial adhesion receptors [8]. Remarkably, ganglioside-like LOS structures expressed on the cell envelope provide serum resistance in humans [60] and play an important role in cellular adhesion on to, invasion into and translocation across intestinal epithelial cells [49,50]. Comparing CRISPR–Cas loci, the presence of sialyltransferase genes and corresponding LOS class and production of sialylated ganglioside-like LOS shows that, in C. jejuni, sialic acid decorations are associated with high resistance to bacteriophages and high virulence (Table 1).

The presence of sialic acid is determined by the sugar decorations on the heptose backbone

Figure 1
The presence of sialic acid is determined by the sugar decorations on the heptose backbone

(A) Addition of one glucose molecule to one of the two LOS backbone heptoses makes the addition of Gal and GalNAc residues possible; sialic acids can be added to Gal residues by the C. jejuni Cst-II transferase enzyme. The C. jejuni Gal-GalNac-NeuAc moieties are also associated with human ganglioside types GM1 or GD1 mimicry [68]; location and numbers of sialic acid residues are the main determinants of mimic type. (B) Addition of glucose residues to both heptose sugars appears to prevent addition of Gal residues and thus sialylation and ganglioside-like LOS expression on the C. jejuni cell envelope [69].

Figure 1
The presence of sialic acid is determined by the sugar decorations on the heptose backbone

(A) Addition of one glucose molecule to one of the two LOS backbone heptoses makes the addition of Gal and GalNAc residues possible; sialic acids can be added to Gal residues by the C. jejuni Cst-II transferase enzyme. The C. jejuni Gal-GalNac-NeuAc moieties are also associated with human ganglioside types GM1 or GD1 mimicry [68]; location and numbers of sialic acid residues are the main determinants of mimic type. (B) Addition of glucose residues to both heptose sugars appears to prevent addition of Gal residues and thus sialylation and ganglioside-like LOS expression on the C. jejuni cell envelope [69].

Table 1
Cst-II-generated ganglioside-like LOS is associated with high resistance to bacteriophages and high virulence

Overview of the CRISPR–Cas locus organization, presence of sialyltransferase genes, LOS class, mimicry to human ganglioside types, capacity to induce GBS and an indication of the virulence and bacteriophage resistance of 20 C. jejuni isolates. The Figure shows the generic subtype II-C CRISPR–Cas locus organization cas9-1-2 (9-1-2); the sialyltransferase genes cst-II and cst-III which are located at a distance of the CRISPR–Cas locus. The Table shows that specifically isolates belonging to LOS classes A and B harbour increased virulence and phage-resistance capabilities [49,50]. *Alterations that affect the function of Cst-II or Cst-III and thereby the expression of ganglioside-like LOS structures. ND, not determined.

Isolate Cas genes Number of repeats cst gene LOS class Mimic GBS induction Virulence Phage resistance 
GB2 9-1-2 cst-II GM1/GD1 Yes High High 
GB3 9-1-2 – cst-II GM1/GD1 Yes Intermediate High 
GB4 9-1-2 – – No Low Intermediate 
GB5 9-1-2 – cst-II* – Yes Low Low 
GB11 9-1-2 – cst-II GM1/GD1 Yes High High 
GB15 9-1-2 ND – – No Low Intermediate 
GB17 9-1-2 ND cst-II* GA1/GM1/GD1 Yes High Intermediate 
GB19 9-1-2 cst-II GM1/GD1 Yes High High 
GB22 9-1-2 cst-II GM1/GD1 Yes Intermediate High 
GB23 9-1-2 cst-II GM2 Yes High Low 
GB29 9-1-2 cst-III* – Yes Low Low 
11168 9-1-2 cst-III GM1/GM2 No Low Intermediate 
706 9-1-2 ND – – No Intermediate Intermediate 
R65 9-1-2 – – No Low Low 
623 9-1-2 ND – – No Low Intermediate 
624 9-1-2 ND – – No Low Intermediate 
9141 9-1-2 ND – – No Intermediate Low 
9146 9-1-2 ND – – No Intermediate Intermediate 
260.94 9-1-2 cst-II ND GM1a Yes High High 
81176 – – cst-II GM2/GM3 No Intermediate High 
Isolate Cas genes Number of repeats cst gene LOS class Mimic GBS induction Virulence Phage resistance 
GB2 9-1-2 cst-II GM1/GD1 Yes High High 
GB3 9-1-2 – cst-II GM1/GD1 Yes Intermediate High 
GB4 9-1-2 – – No Low Intermediate 
GB5 9-1-2 – cst-II* – Yes Low Low 
GB11 9-1-2 – cst-II GM1/GD1 Yes High High 
GB15 9-1-2 ND – – No Low Intermediate 
GB17 9-1-2 ND cst-II* GA1/GM1/GD1 Yes High Intermediate 
GB19 9-1-2 cst-II GM1/GD1 Yes High High 
GB22 9-1-2 cst-II GM1/GD1 Yes Intermediate High 
GB23 9-1-2 cst-II GM2 Yes High Low 
GB29 9-1-2 cst-III* – Yes Low Low 
11168 9-1-2 cst-III GM1/GM2 No Low Intermediate 
706 9-1-2 ND – – No Intermediate Intermediate 
R65 9-1-2 – – No Low Low 
623 9-1-2 ND – – No Low Intermediate 
624 9-1-2 ND – – No Low Intermediate 
9141 9-1-2 ND – – No Intermediate Low 
9146 9-1-2 ND – – No Intermediate Intermediate 
260.94 9-1-2 cst-II ND GM1a Yes High High 
81176 – – cst-II GM2/GM3 No Intermediate High 

Also in N. meningitidis, Neisseria gonorrhoeae, Helicobacter pylori, Haemophilus influenzae and Pasteurella multocida ganglioside-like LOS structures contribute to bacterial pathogenicity [61]. As observed for C. jejuni, ganglioside-like LOS structures on the bacterial cell envelope of H. influenzae appear to play roles in bacteriophage defence and virulence [8,62]. It will be of interest to study whether sialic acid decorations are also involved in bacteriophage defence and virulence in other bacteria belonging to the Type II CRISPR–Cas group as observed for C. jejuni (Figure 2).

Ganglioside-like LOS is linked with increased virulence and bacteriophage resistance in C. jejuni

Figure 2
Ganglioside-like LOS is linked with increased virulence and bacteriophage resistance in C. jejuni

Cst-II-generated ganglioside-like LOS, depicted by a red triangle, has been shown to enhance C. jejuni (green) adherence on to the intestinal epithelial cell line Caco-2 (light pink). This increased adherence went together with increased numbers of C. jejuni bacteria that invaded into, and translocated across, a (polarized) Caco-2 cell monolayer [49,50]. Cst-II-generated ganglioside-like LOS structures (GM1 and GD1) also strongly contribute to resistance of C. jejuni to infection by bacteriophage (orange and brown), probably by interfering with binding of bacteriophage to the C. jejuni cell envelope [59].

Figure 2
Ganglioside-like LOS is linked with increased virulence and bacteriophage resistance in C. jejuni

Cst-II-generated ganglioside-like LOS, depicted by a red triangle, has been shown to enhance C. jejuni (green) adherence on to the intestinal epithelial cell line Caco-2 (light pink). This increased adherence went together with increased numbers of C. jejuni bacteria that invaded into, and translocated across, a (polarized) Caco-2 cell monolayer [49,50]. Cst-II-generated ganglioside-like LOS structures (GM1 and GD1) also strongly contribute to resistance of C. jejuni to infection by bacteriophage (orange and brown), probably by interfering with binding of bacteriophage to the C. jejuni cell envelope [59].

CRISPR–Cas-mediated bacteriophage defence in C. jejuni

Initially, the C. jejuni CRISPR element was used for typing purposes, but it was unknown whether or not the C. jejuni subtype II-C CRISPR–Cas system was functional as a bacteriophage defence system [63]. In the study of Louwen et al. [59] a first glimpse of a potential role of the C. jejuni subtype II-C CRISPR–Cas system in bacteriophage defence was provided. Remarkably, the authors observed, after incubating cas9-negative, cst-II-positive C. jejuni isolates with 29 lytic C. jejuni-infecting bacteriophages, that the CRISPR–Cas system only played a minor role in bacteriophage defence in these C. jejuni isolates that all expressed ganglioside-like LOS structures GM1 and/or GD1 [59]. Curiously, cst-II-positive C. jejuni isolates harboured CRISPR–Cas arrays with significantly reduced CRISPR sizes, accompanied by broad alterations in the cas genes, or even in one strain by complete absence of a CRISPR–Cas system [59]. The correlation between the presence of the cst-II gene and reduced CRISPR sizes and cas gene alterations was of interest, since Cst-II-generated ganglioside-like LOS structures on the C. jejuni cell envelope provided the strongest resistance to lytic C. jejuni-specific bacteriophages [59]. This suggests that in the cst-II-positive strains relief from selection on CRISPR–Cas defence functionality might have allowed shrinkage of the CRISPR repeat numbers and CRISPR array size. Intriguingly, loss of cas9 in cst-II-positive C. jejuni strains did lead to near-complete loss of virulence, and by using human serum from GBS patients were shown to be more profoundly visible to the human immune system [59]. These results provide a hypothetical scenario where a combination of two different bacteriophage defence mechanisms have resulted in C. jejuni isolates that are not only phage-resistant, but also more virulent and linked with post-infectious complications [49,50,53,57,59].

The authors from that study questioned whether the combination of ganglioside-like LOS production and progressively shorter CRISPR repeats was unique for C. jejuni only or whether these features could also be found in other pathogenic bacteria. Combinations of ganglioside-like LOS production and subtype II-C CRISPR–Cas have also been observed in other bacterial pathogens including N. meningitidis and Pasteurellaceae (Haemophilus spp. and P. multocida) [61]. Therefore it was investigated in silico, using the available complete genome sequences representing these pathogens, whether a link between the presence of sialyltransferases and sequence variation in the subtype II-C CRISPR–Cas loci could be identified [59]. Indeed, correlations between the presence of cst-II orthologues and alterations in subtype II-C CRISPR–Cas were detected in N. meningitidis, Haemophilus spp. and P. multocida [59] respectively.

Dual functionalities of bacteriophage defence systems: a blueprint for increased virulence?

In a mathematical model study on the ecology and evolution of CRISPR–Cas systems, Levin [64] concluded that bacteria with a CRISPR–Cas system might have an evolutionary advantage over bacteria employing other types of resistance, i.e. cell envelope resistance, in case the latter comes with a large enough fitness cost. His model also predicted that cell envelope resistance to phages might have an advantage over CRISPR–Cas, again depending on fitness of individual isolates. The outcomes of the models imply that bacteria with bacteriophage cell envelope resistance might replace CRISPR–Cas-bearing lineages if fitness differences are large enough [64]. Furthermore, it was suggested by Levin [64] that bacteria with a CRISPR–Cas system might acquire bacteriophage cell envelope resistance or vice versa, which could result in a population of bacteria with highest fitness compared with the other variants.

The last two outcomes predicted by the models of Levin [64] have been observed in some of the bacterial species within the subtype II-C CRISPR–Cas group. At least some isolates of N. meningitidis, C. jejuni, Haemophilus spp. and P. multocida are characterized by the presence of a CRISPR–Cas system and production of cell envelopes with ganglioside-like LOS structures. It will be of interest to measure whether, within these species, there is a shift at the population level towards an increased proportion of bacteria that harbour both a Type II CRISPR–Cas system and a cell envelope with ganglioside-like LOS structures. Phylogenetic ancestral reconstructions using coalescence theory suggested that C. jejuni isolates producing ganglioside-like LOS originated more recently than enteritis isolates that only used CRISPR–Cas for defence [58], suggesting that cell envelope resistance has been acquired more recently. Such analyses, performed for sufficiently large amounts of isolates sampled as unbiasedly as possible, and sampled at least annually, may show eventually whether there is a tendency in C. jejuni populations to shift towards increasing proportions of ganglioside-like LOS-producing isolates.

Knockout mutagenesis of the C. jejuni subtype II-C CRISPR–Cas marker gene cas9 suggested that there might be a mechanism, at least partially depending on Cas9 function, that is (co-)regulating the production of ganglioside-like LOS structures in clinical C. jejuni isolates obtained from GBS patients [59]. A recent study showed that Cas9, together with two small non-coding RNA species, was involved in degradation of the mRNA encoding an immunogenic lipoprotein that was located at the cell envelope of Francisella novicida [65]. It is intriguing that at least one protein belonging to the Type II CRISPR–Cas bacteriophage defence system is involved in regulation of endogenous protein production or, as suggested by cas9-knockout mutagenesis experiments in C. jejuni, LOS production at the bacterial cell envelope. More evidence is gradually appearing that CRISPR spacers and small non-coding RNA molecules can display high sequence identity with genes encoding proteins involved in bacterial cell envelope synthesis, virulence or stress [31,41,6567], allowing for several intriguing research questions to be posed for Type II CRISPR–Cas harbouring ganglioside-like LOS-expressing bacteria: (i) is production of ganglioside-like LOS as a means of phage defence becoming more widespread in bacteria, for instance in the genera Neisseria, Campylobacter, Haemophilus and Pasteurella?; (ii) does production of ganglioside-like LOS confer an evolutionary advantage on bacteria?; (iii) will the proportions of bacteria that harbour CRISPR–Cas and have the additional capacity to produce ganglioside-like LOS increase at the population level?; (iv) is there further proof for co-regulated dual functionality between the two bacteriophage defence systems CRISPR–Cas and ganglioside-like LOS with respect to phage defence and virulence?; (v) is an interaction between the subtype II-C CRISPR–Cas and ganglioside-like LOS bacteriophage defence systems active during host infection including defence against host-immune response?; and (vi) are the features of LOS sialylation described also operational in LPS (lipopolysaccharide)-expressing bacteria?

Conclusion

In C. jejuni, production of ganglioside-like LOS and alterations in its subtype II-C CRISPR–Cas bacteriophage defence system appear to be correlated in more invasive highly pathogenic isolates. At least in C. jejuni, ganglioside-like LOS and subtype II-C CRISPR–Cas are involved in bacteriophage defence and virulence, suggestive of dual functionality and co-regulation of these two important bacterial processes. Evolutionary models propose that isolates combining both CRISPR–Cas and cell envelope resistance to bacteriophages might experience higher competitive fitness and proportionally increase at the population level. Additional research is required to further elucidate the regulation of production of ganglioside-like LOS at the bacterial cell envelope and the Type II CRISPR–Cas system that may well have evolved different functionalities in bacteria harbouring this system.

CRISPR Evolution, Mechanisms and Infection: A Biochemical Society Focused Meeting held at the University of St Andrews, U.K., 17–19 June 2013. Organized and Edited by Emmanuelle Charpentier (Laboratory for Molecular Infection Medicine Sweden, Sweden), John van der Oost (Wageningen University, The Netherlands) and Malcolm White (University of St Andrews, U.K.).

Abbreviations

     
  • Cas

    CRISPR-associated

  •  
  • CRISPR

    clustered regularly interspaced short palindromic repeats

  •  
  • EPS

    exopolysaccharide

  •  
  • GBS

    Guillain–Barré syndrome

  •  
  • LOS

    lipo-oligosaccharide

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