Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides

Abstract

The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, beta-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin.

FIG. 1.

Role of CPS in the resistance to antimicrobial peptides and proteins. Survival of bacteria (percentage of colony counts of cells not exposed to the agents) with different amounts of HNP-1 (A), HBD1 (B), lactoferrin (C), protamine sulfate (D), and polymyxin B (E). Each point represents the mean and standard deviation of eight samples from four independently grown batches of bacteria, and significant survival differences between 52145 and 52K10 are indicated by asterisks. Symbols: •, K. pneumoniae 52145; ▵, K. pneumoniae 52O21; ○, K. pneumoniae 52K10.

FIG. 2.

Role of CPS and O antigen in the resistance to antimicrobial peptides. (A) Survival of bacteria (percentage of the colony counts of cells not exposed to polymyxin B) after incubation with 1 U of polymyxin B/ml. Error bars display the standard deviation from the mean of three experiments, each one run in duplicate. Also shown are the CPS serotype, the amount of capsule bound to the cell surface, and whether the strains tested express O antigen. (B) Survival of bacteria (percentage of colony counts of cells not exposed to polymyxin B) after incubation with different amounts of polymyxin B. Each point represents the mean and standard deviation of four samples from two independently grown batches of bacteria. Symbols: •, K. pneumoniae USA0352/78 (wild-type strain); ○, K. pneumoniae USA0352/78-3 (CPS mutant).

FIG. 3.

CPS affects the self-promoted pathway. Effect of polymyxin B on the permeability of the OM determined as SDS-induced lysis (A), lysozyme-induced lysis (B), and NPN partition (C). In the absence of polymyxin B, K. pneumoniae 52145 took up more NPN (17,560 ± 200 RLU) than K. penumoniae 52K10 (10,450 ± 200 RLU). Each point represents the mean and standard deviation of four samples from two independently grown batches of bacteria, and significant differences between 52145 and 52K10 are indicated by asterisks. Symbols: •, K. pneumoniae 52145; ○, K. pneumoniae 52K10.

FIG. 4.

CPS affects the binding of polymyxin B to the cell surface. Binding of polymyxin B to purified OMs (A), purified LPSs (B), and bacteria (C) is shown. Each point represents the mean and standard deviation (covered by the symbol) of four samples, and significant differences between 52145 and 52K10 are indicated by asterisks. Symbols: •, K. pneumoniae 52145; ○, K. pneumoniae 52K10.