Mutually constructive roles of Ail and LPS in Yersinia pestis serum survival

Abstract

The outer membrane is a key virulence determinant of gram-negative bacteria. In Yersinia pestis, the deadly agent that causes plague, the protein Ail and lipopolysaccharide (LPS) 6 enhance lethality by promoting resistance to human innate immunity and antibiotics, enabling bacteria to proliferate in the human host. Their functions are highly coordinated. Here we describe how they cooperate to promote pathogenesis. Using a multidisciplinary approach, we identify mutually constructive interactions between Ail and LPS that produce an extended conformation of Ail at the membrane surface, cause thickening and rigidification of the LPS membrane, and collectively promote Y. pestis survival in human serum, antibiotic resistance, and cell envelope integrity. The results highlight the importance of the Ail-LPS assembly as an organized whole, rather than its individual components, and provide a handle for targeting Y. pestis pathogenesis.

Correlating the structure and activity ofY. pestisAil in a bacterial cell envelope

“…Nevertheless, some prominent differences are detectable. These map to sites in the extracellular loops (D23, G60, G66, G105) and near the extracellular membrane-water interface (A11, G28, A48, G92, G94), just below the two clusters of positively charged residues that we identified, recently, as LPSrecognition motifs on opposite poles of the Ail b-barrel [14]: cluster I (R14, K16, K144 and K139), which is tightly localized to the base of the two short extracellular loops EL1 and EL4, and cluster II which occupies a broader region on the barrel surface extending from the base (R27, R51, H95) to the outer extremities (K69, K97, K99) of the two long loops EL2 and EL3. To further examine the side chain conformations of Ail in the bacterial cell envelope, we acquired a 2D 13 C/ 13 C PDSD spectrum with 50 ms mixing at 750 MHz (Fig.…”

“…The outer membrane protein Adhesion invasion locus (Ail) and the lipopolysaccharide (LPS) have co-evolved to enhance microbial resistance to human innate immunity, enabling Y. pestis to produce high-level septicemia in its mammalian hosts [1][2][3][4][5][6][7][8][9][10][11]. Ail and LPS jointly promote evasion of the host immune defenses and adhesion/invasion of host cells [9][10][11], with Ail and LPS mutants exhibiting altered sensitivity to human serum, antibiotics and cell wall stress [12][13][14]. Recently, we identified an LPS-recognition motif on the surface of Ail important for establishing mutually reinforcing Ail-LPS interactions that promote microbial survival in human serum, antibiotic resistance and cell envelope integrity [14].…”

“…Ail and LPS jointly promote evasion of the host immune defenses and adhesion/invasion of host cells [9][10][11], with Ail and LPS mutants exhibiting altered sensitivity to human serum, antibiotics and cell wall stress [12][13][14]. Recently, we identified an LPS-recognition motif on the surface of Ail important for establishing mutually reinforcing Ail-LPS interactions that promote microbial survival in human serum, antibiotic resistance and cell envelope integrity [14]. These features highlight the role of the outer membrane environment as a critical regulator of Ail activity and underscore the importance of characterizing the native Ail-membrane assembly as a whole, rather than its individual components.…”

“…Previous structural studies have relied on purified Ail refolded in detergent micelles, detergent-free lipid nanodiscs or liposomes for X-ray diffraction and NMR [15][16][17][18][19]. We have shown that nanodiscs and liposomes can incorporate Ail with various types of LPS, including native Y. pestis LPS [14,19], but while these detergentfree platforms represent important advances in membrane complexity, they remain distant substitutes for the native outer membrane environment, which is highly anisotropic, heterogeneous, and connected to the cytoskeletal peptidoglycan layer. The emerging field of in-situ NMR [20][21][22][23][24][25] presents new opportunities for examining the structural properties of functional Ail in a native bacterial outer membrane.…”

“…We have shown that nanodiscs and liposomes can incorporate Ail with various types of LPS, including native Y. pestis LPS (14, 19), but even though these detergent-free lipid platforms represent important advances in membrane complexity they remain distant substitutes for the native outer membrane, which is highly anisotropic, heterogeneous, and connected to the cytoskeletal peptidoglycan layer. The emerging field of insitu NMR (20–26) presents new opportunities for examining the structural properties and interactions of Ail in a native bacterial outer membrane.…”