Substitutions in the nonactive site of the passenger domain on the activity of Haemophilus influenzae immunoglobulin A1 protease

Abstract

Immunoglobulin A1 (IgA1) protease is a critical virulence factor of Haemophilus influenzae that facilitates bacterial mucosal infection. This study investigates the effect of iga gene polymorphism on the enzymatic activity of H. influenzae IgA1 protease. The IgA1 protease activity was examined in the H. influenzae Rd KW20 strain and 51 isolates. Genetic variations in iga and deduced amino acid substitutions affecting IgA1 protease activity were assessed. Machine learning tools and functional complementation assays were used to analyze the effects of identified substitutions on the stability and activity of IgA1 protease, respectively. All 51 isolates exhibited similar iga expression levels. No igaB expression was detected. According to comparisons with the reference Rd KW20 strain, four substitutions in the protease domain, 26 in the nonprotease passenger domain, and two in the β-barrel domain were associated with the change in IgA1 protease activity. No substitutions in the catalytic site of IgA1 protease were observed. Logistic regression, receiver operating characteristic curves, Venn diagrams, and protein stability analyses revealed that the substitutions Asn352Lys, Pro353Ala, Lys356Asn, Gln916Lys, and Gly917Ser, which were located in the nonactive site of the passenger domain, were associated with decreases in IgA1 protease activity and stability, whereas Asn914Lys was associated with an increase in these events. Functional complementation assays revealed that the Asn914Lys substitution increased IgA1 protease activity in the Rd KW20 strain. This study identified substitutions in the nonactive site of the passenger domain that affect both the activity and stability of H. influenzae IgA1 protease.

Keywords: Haemophilus influenzae; immunoglobulin A1 protease; virulence factors.

Fig 1

IgA1 protease activities in Haemophilus influenzae isolates. Relative IgA1 protease activity of each isolate compared with the Rd KW20 strain is presented. A change in IgA1 protease activity was defined as a 30% increase or decrease in the mean value compared with that of the Rd KW20 strain. The mean levels of IgA1 protease activity in groups exhibiting a decrease (n = 4), a negligible change (n = 28), and an increase (n = 19) in different dilution folds of culture medium are indicated by red, gray, and blue lines, respectively.

Fig 2

Amino acid substitution rates of the IgA1 protease in the Haemophilus influenzae isolates. (A) Distribution of substitution rates per 10 amino acids of IgA1 protease in the Haemophilus influenzae isolates (n = 51) and (B) phylogenetic characteristics and heatmaps showing the amino acid substitution rates in different regions of H. influenzae IgA1 protease are presented. The sequence of IgA1 protease of the H. influenzae Rd KW20 strain was used as the reference.

Fig 3

Associations of amino acid substitutions with IgA1 protease activity. A Venn diagram for Haemophilus influenzae isolates with different substitutions and IgA1 protease activities is presented. Data are presented as isolate name (relative IgA1 protease activity compared with that of the Rd KW20 strain).

Fig 4

Functional complementation assays. Relative IgA1 protease activity in the Haemophilus influenzae Rd KW20 strain after the expression of IgA1 protease with Asn352Lys-Pro353Ala-Lys356Asn, Gln916Lys-Gly917Ser, or Asn914Lys substitutions is presented. Data were obtained from three independent experiments. P values were obtained from student’s t tests. *P < 0.05; **P < 0.01.