Secondary Acylation of Klebsiella pneumoniae Lipopolysaccharide Contributes to Sensitivity to Antibacterial Peptides

Abstract

Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055⌬lpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055⌬lpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins.Klebsiella pneumoniae is a common cause of nosocomial pneumonia, septicemia, and urinary tract infections; a recent US study found the bacterium to be the third most commonly isolated organism from intensive care wards and the most common species identified in blood cultures (1), with a similar trend reported among European hospitals (2). This rising prevalence combined with the extensive spread of antibiotic-resistant strains, especially extended spectrum ␤-lactamase (ESBL)-producing strains, will drive the search for alternative treatments and/or an effective vaccine against the bacterium.K. pneumoniae is known to express a number of virulence determinants, including a thick polysaccharide capsule. The capsule protects the bacterium from phagocytosis by polymorphonuclear leukocytes (3) and is thought to prevent killing of the bacterium by bactericidal serum factors (4). Other, well studied virulence factors of K. pneumoniae include the pili/fimbriae and lipopolysaccharide (LPS).6 LPS consists of an outer membrane-embedded lipid A molecule, partially conserved inner core carbohydrate structure, attached to an outer core (of which there are at least two variants, conferred by the waa operon (5)), and a repeated polysaccharide or O-antigen (O-Ag). The O-Ag region specifically is thought to play a role in resistance to complement killing (6) and to contribute to bacteremia and lethality during murine pneumonia infections (7) although this contribution is still disputed (8). The comparatively small number of Klebsiella LPS or O-types (9 serotypes) compared with capsule or K-types (Ͼ77 serotypes) suggests that LPS could be utilized for vaccination. However any LPSbased vaccines would need to be detoxified b...

Deciphering tissue-induced Klebsiella pneumoniae lipid A structure

“…S1C). Consistent with previous reports (15,16), the lipid A extracted from Kp52145 grown in lysogeny broth (LB) contained the hexaacylated species m/z 1,824 corresponding to two glucosamines, two phosphates, four R-3-hydroxymyristoyl primary acyl chains, and two myristates (C 14 ) as secondary acyl chains (Figs. 1B and 2).…”

“…Klebsiella LpxM catalyzes the addition of myristate (C 14 ) to the 3′ R-3-hydroxymyristoyl (16). As expected, the lipid A species detected in the Kp52145 lpxM mutant grown in LB were m/z 1,614 and m/z 1,630, which are consistent with the lack of myristate (C 14 ) from species m/z 1,824 and m/z 1,840, respectively (SI Appendix, Fig.…”

“…Ion m/z 1,797 has been previously detected in Klebsiella lipid A and is consistent with two glucosamines, two phosphates, four R-3-hydroxymyristoyl primary acyl chains, and one myristate (C 14 ) and one laureate (C 12 ) as the secondary acyl chains (Fig. 2) (15,16,20,21).…”

“…Species m/z 1,866, not detected in the lipid A from Klebsiella grown in LB, has been previously found in the lipid A from a K. pneumoniae lpxM mutant (16). Klebsiella LpxM catalyzes the addition of myristate (C 14 ) to the 3′ R-3-hydroxymyristoyl (16).…”

Bilayer Properties of Lipid A from Various Gram-Negative Bacteria

“…Three secondary acyl chains are ester-linked to the 2-, 3-, and 3 0 -primary acyl chains, i.e., C2 C 14 (3-OH) and a secondary ester-linked C 16 , C3 C 14 (3-OH), C2 0 C 14 (3-OH) and a secondary ester-linked C 14 , and C3 0 C 14 (3-OH) and a secondary ester-linked C 14 . The hexa-acylated lipid A form is the same as the hepta-acylated form, but there is no secondary C 16 in the 2-position (24,25). In this study, the hepta-and the hexa-acylated lipid A systems are denoted as KP-A and KP-B, respectively.…”