Phasevarions in Haemophilus influenzae biogroup aegyptius control expression of multiple proteins.

IMPORTANCE: Haemophilus influenzae biogroup aegyptius is a human-adapted pathogen and the causative agent of Brazilian purpuric fever (BPF), an invasive disease with high mortality, that sporadically manifests in children previously suffering conjunctivitis. Phase variation is a rapid and reversible switching of gene expression found in many bacterial species, and typically associated with outer-membrane proteins. Phase variation of cytoplasmic DNA methyltransferases has been shown to play important roles in bacterial gene regulation and can act as epigenetic switches, regulating the expression of multiple genes as part of systems called phasevarions (phase-variable regulons). This study characterized two alleles of the ModA phasevarion present in H. influenzae biogroup aegyptius, ModA13, found in non-BPF causing strains and ModA16, unique to BPF causing isolates. Phase variation of ModA13 and ModA16 led to genome-wide changes to DNA methylation resulting in altered protein expression. These changes did not affect serum resistance in H. influenzae biogroup aegyptius strains.

Streptococcus suis Encodes Multiple Allelic Variants of a Phase-Variable Type III DNA Methyltransferase, ModS, That Control Distinct Phasevarions.

Streptococcus suis is a significant cause of bacterial meningitis in humans, particularly in Southeast Asia, and is a leading cause of respiratory and invasive disease in pigs. Phase-variable DNA methyltransferases, associated with restriction-modification (R-M) systems, are a source of epigenetic gene regulation, controlling the expression of multiple genes. These systems are known as phasevarions (phase-variable regulons) and have been characterized in many host-adapted bacterial pathogens. We recently described the presence of a Type III DNA methyltransferase in S. suis, ModS, which contains a simple sequence repeat (SSR) tract within the open reading frame of the modS gene and which differed in length between individual strains. We also observed that multiple allelic variants of the modS gene were present in a population of S. suis isolates. Here, we demonstrate that a biphasic ON-OFF switching of expression occurs in the two most common ModS alleles, ModS1 and ModS2, and that switching is dependent on SSR tract length. Furthermore, we show using single-molecule real-time (SMRT) sequencing that ModS1 and ModS2 are active methyltransferases in S. suis ON-OFF switching of each ModS allele results in the regulation of distinct phasevarions, with the ModS2 phasevarion impacting growth patterns and antibiotic resistance. This is the first demonstration of a phase-variable Type III DNA methyltransferase in a Gram-positive organism that controls a phasevarion. Characterizing the phenotypic effects of phasevarions in S. suis is key to understanding pathogenesis and the development of future vaccines.IMPORTANCEStreptococcus suis is a causative agent of meningitis, polyarthritis, and polyserositis in swine, and it is a major cause of zoonotic meningitis in humans. Here, we investigate epigenetic gene regulation in S. suis by multiple phasevarions controlled by the phase-variable Type III DNA methyltransferase ModS. This is the first characterized example of a Type III R-M system regulating a phasevarion in a Gram-positive organism. We demonstrate that biphasic ON-OFF switching of ModS expression results in differences in bacterial growth and antibiotic resistance. Understanding the effects of ModS phase variation is required to determine the stably expressed antigenic repertoire of S. suis, which will direct and inform the development of antimicrobial treatments and vaccines against this important pathogen.

The Nontypeable Haemophilus influenzae Major Adhesin Hia Is a Dual-Function Lectin That Binds to Human-Specific Respiratory Tract Sialic Acid Glycan Receptors.

NTHi is a human-adapted pathogen that colonizes the human respiratory tract. Strains of NTHi express multiple adhesins; however, there is a unique, mutually exclusive relationship between the major adhesins Hia and HMW1 and HMW2 (HMW1/2). Approximately 25% of NTHi strains express Hia, a phase-variable autotransporter protein that has a critical role in colonization of the host nasopharynx. The remaining 75% of strains express HMW1/2. Previous work has shown that the HMW1 and HMW2 proteins mediate binding to 2-3- and 2-6-linked sialic acid glycans found in the human respiratory tract. Here, we show that the high-affinity binding domain of Hia, binding domain 1 (BD1), is responsible for binding to α2-6-sialyllactosamine (2-6 SLN) glycans. BD1 is highly specific for glycans that incorporate the form of sialic acid expressed by humans, N-acetylneuraminic acid (Neu5Ac). We further show that Hia has lower-affinity binding activity for 2-3-linked sialic acid and that this binding activity is mediated via a distinct domain. Thus, Hia with its dual binding activities functionally mimics the combined activities of the HMW1 and HMW2 adhesins. In addition, we show that Hia has a role in biofilm formation by strains of NTHi that express the adhesin. Knowledge of the binding affinity of this major NTHi adhesin and putative vaccine candidate will direct and inform development of future vaccines and therapeutic strategies for this important pathogen.IMPORTANCE Host-adapted bacterial pathogens like NTHi have evolved specific mechanisms to colonize their restricted host niche. Relatively few of the adhesins expressed by NTHi have been characterized as regards their binding affinity at the molecular level. In this work, we show that the major NTHi adhesin Hia preferentially binds to Neu5Ac-α2-6-sialyllactosamine, the form of sialic acid expressed in humans. The receptors targeted by Hia in the human airway mirror those targeted by influenza A virus and indicates the broad importance of sialic acid glycans as receptors for microbes that colonize the human airway.