Comparative genomics identifies haemin-independent Haemophilus haemolyticus: a formal identification of “Haemophilus intermedius”? Dr. Tegan Harris Senior Research Officer Child Health Division, Menzies

“Haemophilus intermedius” Burbach, 1987 Intermediate between H. influenzae & H. parainfluenzae H. intermedius subsp. intermedius Porphyrin synthesis, ferment sucrose So I’ll start by talking about Haemophilus intermedius, which is an informal species classification given to strains that were thought to be an intermediate between Hi and Hpi, hence their name, as determined by DNA::DNA hybridisation assays which demonstrated H. intermedius had a 60-70% match to each of Hi and Hpi. Two intermedius subspecies defined, but the one we’re most interested in today is subsp. intermedius Porphyrin synthesis and sucrose fermentation  Hpi For the taxonomy freaks, who really enjoy defining species, and identifying new ones, these strains were certainly of interest.

“Haemophilus intermedius” Burbach, 1987 Strains between H. influenzae & H. parainfluenzae H. intermedius subsp. intermedius Porphyrin synthesis, ferment sucrose Strains related to H. haemolyticus Haemin biosynthesis genes hemB, hemE, hemN identified Nørskov-Lauritsen, 2009 More recently, Niels Norskov-Lauritsen’s 2009 publication demonstrated that Haemophilus intermedius are in fact more closely related to H. haemolyticus when more genetic information for comparison of all the Haemophilus species was used. Several haemin biosynthesis genes were identified in these strains, correlating with them not requiring haemin as a growth factor.

“Haemophilus intermedius” Burbach, 1987 Nørskov-Lauritsen, 2014 Strains between H. influenzae & H. parainfluenzae H. intermedius subsp. intermedius Porphyrin synthesis, ferment sucrose Strains related to H. haemolyticus Haemin biosynthesis genes hemB, hemE, hemN identified Nørskov-Lauritsen, 2009 In his 2014 publication, Niels published a Haemophilus species phylogeny, demonstrating that H. intermedius shares a most recent common ancestor with H. haemolyticus, correlating with his findings from 2009.

“Haemophilus intermedius” Year Source Patient 1981 Pleural fluid 69yo 1983 Angular chelitis   1984 Conjunctivitis relapse 4mo Conjunctivitis 3mo 1989 64yo 1992 CSF 1mo 1993 Ascitic fluid Gallbladder 50yo 2000 Epigotitis Urine 2005 12yo 2010 Blood newborn And these H. intermedius are not just a once-off find. Researchers in Sweden have been collecting these strains as part of a growing collection, and currently there are 12 intermedius strains in their collection, all from Sweden, collected over 30 years, from a wide range of clinical specimens/anatomical sites, and from patients of varying age groups

?Haemophilus parainfluenzae Suspected H. parainfluenzae isolates based on phenotype and genotype Haemin independent (X-factor) NAD+ dependent (V-factor) hy4H +ve Out-dated H. haemolyticus speciation assay target that was not species-specific siaT –ve H. influenzae speciation assay target And the reason I’m talking about H. intermedius is because we identified isolates very similar to these within our collection at Menzies. The isolates were originally thought to be H. parainfluenzae, due to only requiring V-factor for growth. And the the genotypic tests originally performed suggested that they were possibly H. haemolyticus.

?H. parainfluenzae  H. haemolyticus These isolates were whole genome sequenced, and using comparative genomics we demonstrated that our suspected H. parainfluenzae isolates, shown here in blue, grouped with the H. haemolyticus isolates, shown in red, rather that H. parainfluenzae in black. Interestingly, rather than spread throughout the tree, these isolates grouped together in what we call a clade, which suggests these isolates share a common ancestor. So at this point, we had identified H. haemolyticus isolates that did not require X-factor, or rather haemin, for growth. A major difference in a key phenotypic test for this species. We were curious to see if these haemin-independent H. haemolyticus were similar to H. intermedius, and through Niels Norskov-Lauritsen we were able to obtain intermedius genomes for comparison.

“H. intermedius” = haemin-independent H. haemolyticus Isolate Origin Year Source 60819_B_Hi1 Australia 2010 BAL 60824_B_Hi4 60971_B_Hi3 2012 60982_B_Hi1 65117_B_Hi3 2011 65151_B_Hi4 839_HINF USA 2013 PN24 Denmark Urine CCUG11096 Sweden 1981 Pleural fluid CCUG15949 1984 Conjunctiva CCUG30218 1992 CSF CCUG31732 1993 Ascitic fluid The analysis just described was repeated, and all 6 of the H. intermedius isolates clustered together with our haemin-independent H. haemolyticus strains shown in blue again, indicating that all of these strains are closely related. Additionally, a publicly available genome from the USA, classified as H. influenzae, also clustered in the haemin-independent clade. So we have these phenotypically unusual, H. haemolyticus isolates, from diverse geographical locations, spanning more than 30 years from isolation, clustering together on this phylogenetic tree. So the next question was, why do these isolates not require haemin for growth? !!! Update figure from SPANDx_Hpi_Hh_020617 !!!

Haemin biosynthesis pathway hemA hemL hemB hemC hemD hemE hemF / hemN / hemZ hemG / hemY And the answer lies in the haemin biosynthesis pathway. Haemin biosynthesis is biological pathway that is common to most forms of life, and consists of 9 main genes. H. Influenzae and H. haemolyticus are two of the very few organisms that are unable to synthesise their own haemin. hemH Schobert, M, & Jahn, D. 2002. JMMB. vol. 4, pg 287-94

Haemin-independent H. haemolyticus hemA hemL hemB hemC hemD hemE hemN x2 hemG & hemY When we investigated our genome assemblies, for the haemin-independent haemolyticus strains, all the genes required for a full haemin biosynthesis pathway were present in their genomes. hemH Schobert, M, & Jahn, D. 2002. JMMB. vol. 4, pg 287-94

Haemin-independent H. haemolyticus All H. haemolyticus hemA hemL hemB hemC hemD hemE hemN hemN x2 hemY hemG & hemY Looking at the “standard” haemolyticus strains, only the the last three genes in the haemin biosynthesis pathway were present. So the next question was, were the haemin biosynthesis genes acquired from a near-neighbour species, such as h. parainfluenzae, or are these haemin-independent H. haemolyticus ancestral, and as the Hhaemolyticus species has evolved, have these genes been lost? hemH hemH Schobert, M, & Jahn, D. 2002. JMMB. vol. 4, pg 287-94

hemA hemL hemB hemC hemD hemE hemN x2 hemG & hemY hemH Schobert, M, & Jahn, D. 2002. JMMB. vol. 4, pg 287-94 hemA hemL hemB hemC hemD hemE hemN x2 hemH hemG & hemY First off, we determined the position of the biosynthesis genes relative to each other within the haemin-independent H. haemolyticus genomes. As you can see in the diagram, the genes are scattered throughout the genome. This is our first indication that the haemin biosynthesis pathway is ancestral to haemolyticus, therefore it has been lost from the rest of the lineage. For this gene arrangement to occur through gene acquisition, each of the genes would have to be acquired individually, and the likelihood of this occurring is very small compared to acquiring all 9 genes in a single block of DNA.

Haemin biosynthesis gene locations Haemin-independent H. haemolyticus Are the genes surrounding the haemin biosynthesis genes the same in all of the isolates? “Standard” H. haemolyticus Does an identical genetic arrangement occur, with an absence of the haemin biosynthesis genes? OR Are entire blocks of DNA, containing haemin biosynthesis genes missing? As the final test of whether these haemin biosynthesis genes were acquired, or lost, from the rest of Hh, we aligned the genomes of the haemin-independent, and “standard” Hh, to see if the haemin biosynthesis gene positions were the same in all of the haemin-independent strains, and if they are, does the same gene arrangement occur in “standard” Hh, or are entire blocks of DNA that contain the haemin biosynthesis genes missing

Haemin biosynthesis gene positions are conserved Haemin-independent H. haemolyticus H. haemolyticus “Standard” Explain this slide more Conserved, or syntenic areas

Haemin biosynthesis gene positions are conserved Haemin-independent H. haemolyticus H. haemolyticus “Standard”

Haemin biosynthesis gene positions are conserved Haemin-independent H. haemolyticus H. haemolyticus “Standard”

Haemin biosynthesis gene positions are syntenic Haemin-independent H. haemolyticus H. haemolyticus “Standard”

Conclusions Haemin-independent H. haemolyticus ?ancestral lineage Haemin biosynthesis lost during evolution of the species X- and V-factor dependence can confound H. haemolyticus identification “Haemophilus intermedius” = haemin-independent H. haemolyticus Classification as a new species is not warranted

Acknowledgements Menzies Child Health Division Funding Collaborators Heidi Smith-Vaughan Jemima Beissbarth Amanda Leach Anne Chang Niels Nørskov-Lauritsen Erin Price Derek Sarovich Collaborators

Haemin biosynthesis genes ≠ H. parainfluenzae When gene sequences were compared to the NCBI nucleotide database, the haemin biosynthesis gene sequences were most similar to H. parainfluenzae. To confirm that these genes were not recently acquired, a concatenated sequence of the haemin biosynthesis pathway genes was created from a H. parainfluenzae strain, and H. parainfluenzae and haemin-independent H. haemolyticus genomes were aligned to this reference. Shown in red, the haemin-independent H. haemolyticus form a distinct cluster compared to the H. parainfluenzae sequences, indicating that the haemin biosynthesis pathway genes in these novel haemolyticus isolates were not recently acquired from H. parainfluenzae.

Haemin biosynthesis gene positions are syntenic in H. haemolyticus Haemin-independent H. haemolyticus hemH

Haemin biosynthesis gene positions are syntenic in H. haemolyticus Haemin-independent H. haemolyticus hemH Haemin-dependent H. haemolyticus

Haemin biosynthesis gene positions are syntenic in H. haemolyticus Haemin-independent H. haemolyticus hemB

Haemin biosynthesis gene positions are syntenic in H. haemolyticus Haemin-independent H. haemolyticus hemB Haemin-dependent H. haemolyticus