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. Author manuscript; available in PMC: 2023 Jun 1.
Published in final edited form as: Curr Opin Infect Dis. 2022 Jun 1;35(3):188–195. doi: 10.1097/QCO.0000000000000830

Contributions of Yersinia pestis outer membrane protein Ail to plague pathogenesis

Anna M Kolodziejek 1, Carolyn J Hovde 1, Scott A Minnich 1,#
PMCID: PMC9186061  NIHMSID: NIHMS1798733  PMID: 35665712

Abstract

Purpose:

Pathogenic Yersinia have been a productive model system for studying bacterial pathogenesis. Hallmark contributions of Yersinia research to medical microbiology are legion and include: (i) the first identification of the role of plasmids in virulence, (ii) the important mechanism of iron acquisition from the host, (iii) the first identification of bacterial surface proteins required for host cell invasion, (iv) the archetypical type III secretion system, and (v) elucidation of the role of genomic reduction in the evolutionary trajectory from a fairly innocuous pathogen to a highly virulent species.

Recent findings:

The outer membrane protein Ail (attachment invasion locus) was identified over 30 years ago as an invasin-like protein. Recent work on Ail continues to provide insights into Gram-negative pathogenesis. This review is a synopsis of the role of Ail in invasion, serum resistance, outer membrane stability, thermosensing, and vaccine development.

Summary:

Ail is shown to be an essential virulence factor with multiple roles in pathogenesis. The recent adaptation of Y. pestis to high virulence, which included genomic reduction to eliminate redundant protein functions, is a model to understand the emergence of new bacterial pathogens.

Keywords: Yersinia pestis, plague pathogenesis, Ail, serum resistance, outer membrane stability

INTRODUCTION

Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis are three species within this genus causing zoonotic diseases. The first two are enteropathogens that cause mild, localized, self-limiting intestinal infections. In contrast, Y. pestis causes plague, a systemic disease with the potential to progress to a pneumonic form and human-to-human airborne transmission (1). Y. pestis has restricted host niches and cycles between wild rodents and a flea vector. Occasionally, it is transmitted to humans and other mammals by the bite of an infected flea or by direct contact with a diseased animal (2). Plague most likely originated in Asia and spread globally, reaching North America at the turn of the late 19th century (3, 4). Current endemic sites are in Asia, Africa, and the Americas (5).

It is well accepted that Y. pestis evolved from Y. pseudotuberculosis around 5,700-6,000 years ago (5–8). Genomes of the two species share ~97% DNA sequence homology (1). The few differences reflect mutations, deletions, rearrangements, and gene acquisition by lateral gene transfer that drove Y. pestis into a biphasic life cycle: colonization of the insect midgut and immune evasion in mammals (1, 9). Acquisition of virulence and flea colonization factors was accompanied by a significant ~13% genome reduction from the progenitor, Y. pseudotuberculosis. These combined genomic change mechanisms resulted in differential gene expression, changes in structural composition, and metabolic restrictions (5, 10, 11).

Two virulence-associated loci, the pCD1 plasmid (pYV in Y. pseudotuberculosis and Y. enterocolitica) and chromosomally encoded ail (attachment invasion locus), are present in all three Yersinia spp. that cause mammalian diseases. The virulence plasmids encode a type three secretory system (T3SS) that delivers cytotoxic Yersinia outer proteins (Yops) into host cells (12). Ail is a 17.5 kDa outer membrane (OM) protein mediating attachment to and invasion of host cells, and serum resistance (13, 14). Ail also regulates OM thermostability in Y. pestis (15). Y. pseudotuberculosis and Y. enterocolitica both have additional virulence factors that overlap with Ail function, but due to genomic reduction in Y. pestis, Ail alone, accounts for the Y. pestis phenotypes. Functional ail is present in the genomes of all Y. pestis strains, including representatives of the oldest lineages circulating today. Y. pestis Ail shares high homology with Ail from Y. enterocolitica and Y. pseudotuberculosis (approximately 70 and 99%, respectively). It is also highly conserved within Y. pestis sp. (Fig. 1A) (13, 16).

FIGURE 1. Primary and tertiary structure of Ail from Yersinia pestis KIM.

FIGURE 1.

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Ail is depicted as a mature protein in the OM and amino acid are numbered from the cleaved N-terminus. Extracellular loops are indicated as EL1, EL2, EL3, and EL4. A) Primary amino acid sequence residues are shown in color with gray as coil-forming and blue as β-strand-forming. Asterisks denote changes in amino acid sequence among Y. pestis strains, including the serine insertion at E108_S109insS. B) The predicted Ail structure was generated using the deep learning-based modelling program, RoseTTAFold, and presented with UCSF Chimera (66, 67). Front and back orientations are shown. Orange residues indicate LOS recognition motifs denoted as Cluster I and Cluster II. Cluster I is formed by residues R14, K16, K144, and K139. Cluster II is divided in two regions: the outer cluster (K69, K97, K99) and base cluster (R27, R51, Y71, H95) (34). Underlined residues indicate heparin binding sites HBSI (K69-H95-K97-K99) and HBS II (R14-K16-K21) (52). Boxed tryptophan residues are involved in folding (W41) and stabilization of the folded protein (W148) (59). Residues F54-F104-D67-F68, E17-G96 combined with Cluster II determine serum resistance (34, 38, 60). Residues F54 and F104 are required for vitronectin, factor H, and C4BP binding (38). Residues F54, S102, and F104 contribute to cell adhesion. Residue F54 is critical for laminin binding. Combinations of F54-F104 and F54-S102-F104 and F68 residue are required for fibronectin binding (52, 60). The hydrophobic belt defines the membrane spanning region exposed to the hydrophobic core of the lipid bilayer (52).

This review focuses on the multivalent functions of Ail in Y. pestis pathogenesis and physiology and compares it with Ail in enteropathogenic yersiniae. Particularly, it addresses the non-redundant role of Ail in pathogenesis, including determination of T3SS efficacy, dictated by Y. pestis genome entropy.

AIL EXPRESSION AND Y. PESTIS ADAPTATION TO A BIPHASIC LIFE CYCLE

Ail expression in Y. pestis and food-borne Yersinia sp. is optimal at 37° C (17–19). The protein constitutes approximately 30% of the Y. pestis OM proteome and is the most transcribed protein at mammalian host temperature (20–22). It is noteworthy that high Ail expression is also maintained at ambient temperature by Y. pestis while significantly reduced in enteropathogenic Yersinia (13, 17–19, 21–24). Comparing Y. pestis and Y. pseudotuberculosis ail expression at ambient temperature is also different during flea colonization (25). Y. pestis ail is expressed at a higher level than ail from a flea-transmissible Y. pseudotuberculosis mutant strain. Even though Ail is not required for flea infection or biofilm blockage in the flea (14), its upregulation may predispose Y. pestis to resist complement components during flea-bite transmission (25). This hypothesis is supported by the sharp decline in Ail expression observed only at low temperatures (6° C), consistent with the winter season during which rodents hibernate, fleas are dormant, and there are decreased transmission rates (14). Interestingly, repeated Y. pestis passaging in the laboratory can also result in loss of Ail expression (our observations and 26). Frameshift mutations or transposon insertions in ail occur in laboratory strains but have not been reported in any wild-type isolates (main or non-main spp.) (26). This indicates that Ail expression is required for Y. pestis maintenance and persistence in nature. Activation of ail in Y. pseudotuberculosis is controlled by RNA thermometers (17). The mechanisms of ail differential temperature-regulation in Y. pestis is not known and may be involved in its transition to an arthropod-borne pathogen.

ROLE OF AIL IN PATHOGENESIS AND VIRULENCE

To maintain a biphasic life-cycle, high Y. pestis numbers are required in mammalian blood to ensure transmission back to the flea (2). Serum resistance, inhibition of the inflammatory response, and selective injection of cytotoxic Yops into immune cells are essential to evade mammalian innate immunity and establish infection (27–29). All ail-negative, serum sensitive strains are cleared efficiently in rats by inducing both innate and adaptive immune responses that include the inflammatory response with recruitment of polymorphonuclear leukocytes to the infection site and proximal lymph nodes (29, 30). Ail-mediated phenotypes contributing to Y. pestis pathogenesis are described below.

Ail-mediated serum resistance: pathogenesis

Resistance to the bactericidal activity of serum complement is the most prominent role of Ail in Y. pestis (13, 14, 29). In contrast, enteric Yersinia express two additional complement resistance factors: YadA and the O-chain in LPS. Both of these factors have been lost in in Y. pestis. The plasmid-borne yadA is mutated, and the O-antigen synthesis gene cluster is truncated by mutation (31–33). Ail specifically interacts with truncated LPS, referred to as lipo-oligosaccharide (LOS) (34). Mutations in ail coding for amino acids that interact with LOS or further truncation of the LOS outer core results in serum sensitively (29, 34).

Due to transmission between the insect and mammal, and the systemic character of the disease, Y. pestis contact with blood occurs over a broad range of temperatures. Y. pestis is resistant to complement when grown at 37° C (mammalian host), 28° C (insect vector), but not at 6° C (winter season with decreased insect activity) and correlates with Ail expression patterns. Complement-killing of ail-negative strains is observed with human, rat, goat, sheep, rabbit, and guinea pig sera, but not with mouse sera (13, 14, 35). It is well known that murine sera have limited bactericidal activities against many microorganisms (13, 14, 35). Consequently, experimental Y. pestis infections in mice and rats have significant differences and show that Ail mediates additional host cell adherence and invasion properties. All studies using a rat model for bubonic, pneumonic, and septicemic plague, show attenuation of ail-negative strains (29, 30). This highlights the critical role of Ail-mediated serum resistance in the disease. In contrast, this is not the case in murine infection. There is much inconsistency among murine studies that is dependent on the route of infection (intranasal, subcutaneous, or intravenous), (29, 30, 36, 37). Use of different Y. pestis strains (Y. pestis KIM5, Y. pestis CO92, or Y pestis KIMD27) may contribute to the observed discrepancies, as well.

Ail-mediated serum resistance: Ail interactions with C4BP, factor H, C4b, C3b, and vitronectin

Ail interacts with complement components and regulators to confer serum resistance in Yersinia spp. (38). Interactions of Ail with complement inhibitor C4b-binding protein (C4BP) and factor H are confirmed in Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis (38–43). Binding of C4BP by Ail is specific and involves C-terminus residues on C4BP: complement control protein (CCP) domains 6 and 8 for both Y. pseudotuberculosis and Y. pestis Ail (39, 40). Y. pestis Ail also binds vitronectin, C3b, and C4b and these interactions have not been studied in enteropathogenic Yersinia (39, 44).

Factor H and C4BP are the most important fluid-phase regulators of the alternative, classical, and lectin complement pathways. Both are utilized by many pathogens to circumvent complement (45–47). Factor I is a serine protease that inactivates C3b and C4b components by cleavage. Ail recruits factor H and C4BP to interact with bacterial surface-bound C3b and C4b, respectively. Interactions between factor H and C3b, and/or C4BP and C4b promotes binding of factor I to the complexes. Factor I then cleaves C3b and C4b to iC3b and iC4b. This inactivation represses the complement cascade on the bacterial surface (38, 39, 47). In addition to these covalent interactions, Y. pestis Ail binds noncovalently to C4b and C3b (39). C4b with Ail can be inactivated by factor I. Whether C3b with Ail is cleaved and inactivated by a similar mechanism employing factor H or factor I is yet to be determined (39).

Vitronectin is known to inhibit the complement cascade at the terminal complement complex formation (TCC). It blocks membrane binding of C5b complexes and prevents C9 polymerization (48). Y. pestis Ail binds the C-terminal domain of vitronectin and facilitates its cleavage by bacterial Pla protease (44, 49). The significance of these interactions is yet to be determined.

Ail-mediated cell adherence and invasion

Ail is a primary adhesin in Y. pestis. It acts independently from other Y. pestis proteins (29, 37). Two other major adhesins in enteric Yersinia, invasin and YadA, are inactivated by mutations in Y. pestis. Ail confers binding to and invasion of host cells and contributes to efficient targeting and Yop delivery to host cells, including to neutrophils (13, 37, 50–53). These activities are inhibited if the O-antigen extends beyond the LPS core in either Y. pestis or Y. pseudotuberculosis (29, 54). In contrast, truncation of the LPS inner core in an E. coli model, decreases Y. pestis Ail-conferred host cell invasion but does not affect adherence (29). Ail mediates Y. pestis adherence and facilitates Yop injection to host cells by direct interaction with extracellular matrix (ECM) proteins bound to a receptor on a host cell. Ail interacts with laminin (LG4-5 fragment), fibronectin (9FNIII fragment), vitronectin, and heparan sulfate proteoglycans, but not with collagen (44, 49, 52, 53, 55).

Ail-mediated adherence and host cell invasion during Y. pestis lung infections has varied results. Studies by Eichelberger et al. suggest that early Y. pestis adherence to host cells during primary pneumonic plague is Ail-independent (56). Also, the loss of Ail in Y. pestis CO92 does not lower the LD50 dose in a murine model of pneumonic infection (29). However, recent findings by Zheng et al. indicate that Y. pestis KIMD27, an ail-negative strain, is attenuated in intranasal and orogastric challenge in mice (36). In Y. pseudotuberculosis, Ail is non-redundant during early colonization of the lungs by a special variant that can colonize and rapidly disseminate. However, it is redundant with YadA for specific targeting of neutrophils, persistence in and dissemination from lungs at later stages of infections (51).

Deletion of Ail attenuates Y. pestis in a murine bubonic plague model. Infection with ail-negative strains produces high numbers of neutrophils at the infection site and proximal lymph nodes (30). This suggests that Ail inhibits neutrophil recruitment and/or induces their death at these sites. The latter correlates with Ail-conferred adhesion and injection of cytotoxic Yops into host cells in vitro (37, 53). The role of these Ail-mediated phenotypes in murine septicemic models is not resolved(36, 37).

TEMPERATURE-DEPENDENT CONTRIBUTION OF AIL TO Y. PESTIS HEAT SHOCK RESPONSE AND OM STABILITY

Until recently Ail was only associated with virulence; however, current data show that it also plays unique roles in thermal signaling and maintaining OM stability at elevated temperature in Y. pestis (15, 34). Deletion of ail suppresses induction of rpoE and rpoH heat shock response sigma factors when cells are grown at 37° C (15). This shows Ail is a key signaling component of the Y. pestis thermoregulatory system. Transmission from the flea to a warm-blooded mammal is a significant temperature change. The Ail-dependent induction of the heat shock response during vector to host transitions has not been studied.

Y. pestis Ail is essential for maintaining OM lipid asymmetry and lipid homeostasis at 37° C (15). Under physiological conditions, the lipid content of the OM of Gram-negative bacteria is asymmetric, with LPS/LOS molecules occupying the outer leaflet and phospholipids (PL) occupying the inner leaflet. Interactions between Ail and LOS in Y. pestis mutually determine their structures; LOS provides the environment for an extended conformation of Ail at the membrane surface and Ail causes localized thickening and reduction of LOS fluidity (34). Deletion of Ail causes an aberrant flow of PL into the OM outer leaflet. It also activates phospholipase A (PldA), an OM enzyme with broad substrate specificity that removes PL from the OM outer leaflet by hydrolysis. (15). This loss of lipid asymmetry changes Y. pestis cell morphology. Cellular components are released during logarithmic phase growth and plasmolysis and lysis occurs as cells transition into stationary phase growth. This morphological change is suppressed by mutations in pldA, suggesting that ail-negative cells are losing membranes faster than new PL can be synthesized. Growth with substrates promoting lipid biosynthesis, such as glycerol, or growth on glucose that suppresses pldA expression, stabilizes OM lipid A symmetry in ail mutants. Similarly, addition of calcium ions, interacting with negatively charged LOS molecules and anionic PL, have the same effect (15). This Ail-mediated OM stabilization also facilitates Caf1 capsule assembly, a bacterial surface structure that prevents phagocytosis (15, 57).

Detailed mechanisms of temperature-sensitive lysis are not understood, but it is known that the process is regulated indirectly by the rpoE regulon (15). Also, lysis of ail-negative mutants at 37° C, but not at 28° C, correlates with the transition from LOS hexaacylation to tetraacylation at this temperature. The observed increase in Ail expression at 37° C may compensate for the change in lipid fluidity associated with the acylation state of Lipid A. The Ail contribution to OM stabilization at higher temperature may be critical in pathogenesis because the transition to tetraacylated LOS allows bacteria to escape Toll-like receptor-4 (TLR-4) detection.

AIL MOLECULAR STRUCTURAL AND OM STABILITY, SERUM RESISTANCE, ADHESION, YOP DELIVERY, AND AUTOAGGREGATION FUNCTIONS

Ail assembly in the OM and OM stability

Ail forms an 8-stranded, antiparallel β-barrel with four extracellular and three intracellular loops (Fig.1B) (52, 58). Residue W148 maintains barrel stability and residue W41 drives barrel refolding but does not contribute to the subsequent stability of the folded protein (59). Y. pestis LOS and Ail interact specifically in the membrane with mutually stabilizing effects (34). Two Ail LOS-recognition motifs, Cluster I and II, are present. Cluster I localizes to the base of the extracellular loops 1 and 4 (R14, K16, K144, K139). Cluster II covers residues on the extracellular loops 2 and 3 (outer cluster: K69, K97, K99) and their base on the barrel surface (base cluster: R27, R51, Y71, H95) (Fig.1B). Both Clusters form hydrogen bonds with the phosphate and hydroxyl groups of LOS. Cluster I primarily interacts with tetraacylated lipid IVA and Cluster II interacts with the core oligosaccharide groups (34). Mutations, either in the base or outer components of Cluster II, destabilize the OM and results in loss of cell integrity (34). Also, mutations in base Cluster II sensitizes Y. pestis to polymyxin B, a cationic antibiotic that binds to LOS and induces OM permeability (15, 34).

Ail-mediated serum resistance

Cluster II LOS-recognition motif (base and outer Cluster) is required for serum resistance (34). In addition, the combination of F54-F104-D67-F68 or E17-G96 residues determine serum resistance (38, 60). Recruitment of three complement regulatory factors: vitronectin, factor H, and C4BP involve F54 and F104 (Fig.1B). However, these residues contribute moderately and are not essential for serum resistance (38). This suggests that Ail-mediated serum resistance in Y. pestis involves multiple mechanisms with some not yet identified.

Ail-mediated cell and ECM adhesion

Extracellular loops 1, 2 and 3 participate in Ail-mediated interaction of Y. pestis with host cells and the ECM (52, 60). Residues F54, S102, and F104 contribute to cell adhesion. Residue F54 is critical for laminin binding. Combinations of F54-F104 and F54-S102-F104 and F68 residues are required for fibronectin binding. For heparin binding, heparin binding sites (HBS) were identified. Residues K69-H95-K97-K99 (loops 2 and 3) form HSB-I and R14-K16-K21 (loop 1) form HBS-II (52) (Fig.1B). Amino acid substitutions found in Ail of some Y. pseudotuberculosis strains (E17D and F100V) or the less virulent Y. pestis spp. (F100V) have a modest defect on Ail-mediated cell adhesiveness and invasiveness, and fibronectin binding properties (54).

Ail-mediated Yop delivery

Residues required for cell adhesion and ECM binding (F54-F104, and F54-S102-F104) are also required for Ail-mediated Yop delivery (Fig.1B). This suggests that fibronectin and laminin are bridging molecules between Ail and host cells for T3SS Yop delivery (60).

Ail-mediated autoaggregation

Y. pestis autoaggregation requires Ail-Ail interaction between cells that occurs between extracellular loops 2 and 3. It is determined dominantly by F104 and augmented by F54 residues (60) (Fig.1B).

VACCINE POTENTIAL OF AIL

The potential use of Y. pestis in biowarfare, the appearance of drug resistant strains, and recent outbreaks of urban pneumonic plague in Madagascar, warrant efforts to develop an efficacious and safe vaccine. Good vaccine candidates include Ail as a subunit or live-ail-attenuated strains. Recombinant Ail is immunogenic and provides partial protection against bubonic and pneumonic plague in mice and rats, respectively (61). Immunization with the ail-negative strains induce robust inflammatory response in lymphoid tissues (30, 62). A two-dose vaccine with an attenuated strain lacking Ail and two other proteins, Brown lipoprotein and lipid A myristoyltransferase, provides full protection against pneumonic plague in mice and rats (63, 64). Combinatorial immunization with the same strain and adenovirus vector expressing T3SS needle subunit protein YscF, capsular antigen F1, and V antigen LcrV (intranasal route) provides the same level of protection in mice with a one-dose regimen (65).

CONCLUSION

This review compiles the current understanding of the multivalent functions of the Y. pestis OM protein Ail and also identifies areas for further study. Ail is common for all pathogenic Yersinia and plays an integral role in Y. pestis pathogenesis and the preservation of OM lipid asymmetry. It is well accepted that Y. pestis emerged from its innocuous progenitor, Y. pseudotuberculosis, through genomic reduction and lateral gene transfer. This process eliminated proteins with redundant Ail functions and elevated Ail to a central role in plague pathogenesis. This is a model for understanding the emergence of highly pathogenic bacteria.

Key points.

  • Emergence of Y. pestis from an enteric predecessor included elimination of proteins with redundant Ail functions and elevated Ail to a central role in plague pathogenesis

  • Resistance to the bactericidal activity of serum complement is a prominent role of Ail in Y. pestis

  • Ail is a primary adhesin in Y. pestis and mediates efficient T3SS delivery of cytotoxic Yops into host cells

  • Ail plays unique roles in thermal signaling and maintaining OM stability at elevated temperature in Y. pestis

  • Good vaccine candidates include the Ail subunit or live-ail-attenuated strains

ACKNOWLEDGMENTS

This project was supported, in part, by the USDA National Institute for Food and Agriculture, Hatch projects IDA01574 (CJH and AK) and IDA01406 (SAM and AK), the National Institute of General Medical Sciences of the National Institutes of Health Grants #P20GM103408, #U54-AI057141 and the University of Idaho Agriculture Experiment Station.

No funding was received for this work.

Footnotes

Conflicts of interests

None

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