Overlapping and complementary oxidative stress defense mechanisms in nontypeable Haemophilus influenzae.

The Gram-negative commensal bacterium nontypeable Haemophilus influenzae (NTHI) can cause respiratory tract diseases that include otitis media, sinusitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During colonization and infection, NTHI withstands oxidative stress generated by reactive oxygen species produced endogenously, by the host, and by other copathogens and flora. These reactive oxygen species include superoxide, hydrogen peroxide (H2O2), and hydroxyl radicals, whose killing is amplified by iron via the Fenton reaction. We previously identified genes that encode proteins with putative roles in protection of the NTHI isolate strain 86-028NP against oxidative stress. These include catalase (HktE), peroxiredoxin/glutaredoxin (PgdX), and a ferritin-like protein (Dps). Strains were generated with mutations in hktE, pgdX, and dps. The hktE mutant and a pgdX hktE double mutant were more sensitive than the parent to killing by H2O2. Conversely, the pgdX mutant was more resistant to H2O2 due to increased catalase activity. Supporting the role of killing via the Fenton reaction, binding of iron by Dps significantly mitigated the effect of H2O2-mediated killing. NTHI thus utilizes several effectors to resist oxidative stress, and regulation of free iron is critical to this protection. These mechanisms will be important for successful colonization and infection by this opportunistic human pathogen.

HrrF is the Fur-regulated small RNA in nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) are Gram-negative commensal bacteria that reside in the nasopharynx. NTHi can also cause multiple upper and lower respiratory tract diseases that include sinusitis, conjunctivitis, bronchitis, and otitis media. In numerous bacterial species the ferric uptake regulator (Fur) acts as a global regulator of iron homeostasis by negatively regulating the expression of iron uptake systems. However in NTHi strain 86-028NP and numerous other bacterial species there are multiple instances where Fur positively affects gene expression. It is known that many instances of positive regulation by Fur occur indirectly through a small RNA intermediate. However, no examples of small RNAs have been described in NTHi. Therefore we used RNA-Seq analysis to analyze the transcriptome of NTHi strain 86-028NPrpsL and an isogenic 86-028NPrpsLΔfur strain to identify Fur-regulated intergenic transcripts. From this analysis we identified HrrF, the first small RNA described in any Haemophilus species. Orthologues of this small RNA exist only among other Pasteurellaceae. Our analysis showed that HrrF is maximally expressed when iron levels are low. Additionally, Fur was shown to bind upstream of the hrrF promoter. RNA-Seq analysis was used to identify targets of HrrF which include genes whose products are involved in molybdate uptake, deoxyribonucleotide synthesis, and amino acid biosynthesis. The stability of HrrF is not dependent on the RNA chaperone Hfq. This study is the first step in an effort to investigate the role small RNAs play in altering gene expression in response to iron limitation in NTHi.

Draft Genome Sequence of the Clinical Isolate Acinetobacter nosocomialis Strain M2.

We report the 3.78-Mbp high-quality draft assembly of the genome from a clinical isolate of Acinetobacter nosocomialis called strain M2 (previously known as Acinetobacter baumannii strain M2).

Construction of non-polar mutants in Haemophilus influenzae using FLP recombinase technology.

BACKGROUND: Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium that causes otitis media in children as well as other infections of the upper and lower respiratory tract in children and adults. We are employing genetic strategies to identify and characterize virulence determinants in NTHi. NTHi is naturally competent for transformation and thus construction of most mutants by common methodologies is relatively straightforward. However, new methodology was required in order to construct unmarked non-polar mutations in poorly expressed genes whose products are required for transformation. We have adapted the lambda red/FLP-recombinase-mediated strategy used in E. coli for use in NTHi. RESULTS: A cassette containing a spectinomycin resistance gene and an rpsL gene flanked by FRT sites was constructed. A PCR amplicon containing 50 base pairs of DNA homologous to the 5' and 3' ends of the gene to be disrupted and the cassette was generated, then recombineered into the target NTHi gene, cloned on a plasmid, using the lambda recombination proteins expressed in E. coli DY380. Thus, the gene of interest was replaced by the cassette. The construct was then transformed into a streptomycin resistant NTHi strain and mutants were selected on spectinomycin-containing growth media. A plasmid derived from pLS88 with a temperature sensitive replicon expressing the FLP recombinase gene under the control of the tet operator/repressor was constructed. This plasmid was electroporated into the NTHi mutant at the permissive temperature and FLP expression was induced using anhydrotetracycline. The recombinase recognizes the FRT sites and eliminates the antibiotic cassette by site-specific recombination, creating the unmarked non-polar mutation. The plasmid is cured by growth of cells at the restrictive temperature. CONCLUSION: The products of the genes in the NTHi pilABCD operon are required for type IV pilus biogenesis and have a role in transformation. We demonstrated the utility of our methodology by the construction of a non-polar pilA mutation in NTHi strain 2019 and complementation of the mutation with a plasmid containing the pilA gene. Utilization of this approach allowed us to readily generate unmarked non-polar mutations in NTHi genes.

The PilA protein of non-typeable Haemophilus influenzae plays a role in biofilm formation, adherence to epithelial cells and colonization of the mammalian upper respiratory tract.

We recently described the expression of type IV pili (Tfp) by non-typeable Haemophilus influenzae (NTHI), a common respiratory tract pathogen. Prior to that report, Tfp were not thought to be produced by NTHI as they are not observed on NTHI when grown on chocolate agar or other commonly used growth media. To further characterize growth conditions permissive for the expression of NTHI Tfp, as well as determine their role in colonization and virulence, we transformed an NTHI otitis media isolate with a reporter plasmid containing the lux gene cluster driven by the pilA promoter. Transcription from the pilA promoter was demonstrated under a variety of in vitro growth conditions and, importantly, by ex vivo imaging of luciferase-producing NTHI in infected chinchillas. Luciferase-producing NTHI were also identified within a biofilm formed by NTHI in vivo. We further demonstrated a role for NTHI PilA in adherence to human respiratory epithelial cells, in colonization of the chinchilla respiratory tract as well as a requirement for PilA in biofilm development, both in vitro and in vivo. Collectively, our data demonstrate that NTHI express PilA in vivo, and that PilA plays an important role in the pathogenesis of an upper respiratory tract infection induced by NTHI.

The OxyR regulon in nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium and a common commensal organism of the upper respiratory tract in humans. NTHi causes a number of diseases, including otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During the course of colonization and infection, NTHi must withstand oxidative stress generated by insult due to multiple reactive oxygen species produced endogenously by other copathogens and by host cells. Using an NTHi-specific microarray containing oligonucleotides representing the 1821 open reading frames of the recently sequenced NTHi isolate 86-028NP, we have identified 40 genes in strain 86-028NP that are upregulated after induction of oxidative stress due to hydrogen peroxide. Further comparisons between the parent and an isogenic oxyR mutant identified a subset of 11 genes that were transcriptionally regulated by OxyR, a global regulator of oxidative stress. Interestingly, hydrogen peroxide induced the OxyR-independent upregulation of expression of the genes encoding components of multiple iron utilization systems. This finding suggested that careful balancing of levels of intracellular iron was important for minimizing the effects of oxidative stress during NTHi colonization and infection and that there are additional regulatory pathways involved in iron utilization.

Demonstration of Type IV pilus expression and a twitching phenotype by Haemophilus influenzae.

Haemophilus influenzae is considered a nonmotile organism that expresses neither flagella nor type IV pili, although H. influenzae strain Rd possesses a cryptic pilus locus. We demonstrate here that the homologous gene cluster pilABCD in an otitis media isolate of nontypeable H. influenzae strain 86-028NP encodes a surface appendage that is highly similar, structurally and functionally, to the well-characterized subgroup of bacterial pili known as type IV pili. This gene cluster includes a gene (pilA) that likely encodes the major subunit of the heretofore uncharacterized H. influenzae-expressed type IV pilus, a gene with homology to a type IV prepilin peptidase (pilD) as well as two additional uncharacterized genes (pilB and pilC). A second gene cluster (comABCDEF) was also identified by homology to other pil or type II secretion system genes. When grown in chemically defined medium at an alkaline pH, strain 86-028NP produces approximately 7-nm-diameter structures that are near polar in location. Importantly, these organisms exhibit twitching motility. A mutation in the pilA gene abolishes both expression of the pilus structure and the twitching phenotype, whereas a mutant lacking ComE, a Pseudomonas PilQ homologue, produced large appendages that appeared to be membrane bound and terminated in a slightly bulbous tip. These latter structures often showed a regular pattern of areas of constriction and expansion. The recognition that H. influenzae possesses a mechanism for twitching motility will likely profoundly influence our understanding of H. influenzae-induced diseases of the respiratory tract and their sequelae.

Yersinia pseudotuberculosis produces a cytotoxic necrotizing factor.

Cell extracts from Yersinia pseudotuberculosis induced multinucleation in HEp-2 cells in a manner similar to the effect caused by Escherichia coli cytotoxic necrotizing factor (CNF). The activity was not dependent on the Yersinia 70-kb virulence plasmid, and the activity was not inhibited by antibodies capable of neutralizing E. coli CNF type 1. The nucleotide sequence of the Yersinia cnf gene was 65.1% identical to the E. coli cnf gene.