One Ring To Bind Them All: Is Heme Biosynthesis A Factor InWolbachia-Filarial Nematode Endosymbiosis?

Introduction:Transmitted by insect vectors, human nematode-based filariasis causes debilitating diseases affecting nearly 150 million people with 1.2 billion individuals at riskin 80 countries. Genomic sequencing has revealed that many human filarial nematodes, such as Wuchereria bancrofti, Brugia malayi and B. timori, (causativeagents of lymphatic filariasis (LF)) and Onchocerca volvulus (causative agent of onchocerciasis (river blindness)) contain the obligate endosymbiont, Wolbachia.Genome sequencing of B. malayi (Bm) and its Wolbachia (wBm) identified a number of metabolites implicated in the host-endosymbiont interaction, one ofwhich was heme, a co-factor in a number of enzymes and essential to many biological processes. Although the Bm genome encodes a functional ferrochelatasegene (the final step in the heme biosynthetic pathway and a product of lateral gene transfer), like other nematodes, it is incapable of synthesizing heme.However, the wBm genome contains a functional heme synthesis pathway, leading to the hypothesis that wBm may supply Bm with heme.

Our laboratory is exploiting the use of biochemistry, cytology and Next-Generation sequencing to further investigate patterns of Bm and wBm heme trafficking.

Acknowledgements: B. malayi adults and mf were from TRS Labs, Inc (Athens, GA). This work was funded by New England Biolabs.

Glycine + Succinyl-CoA

5’-aminolevulinate

Porphobilinogen

Uroporphyrinogen III

Coproporphyrinogen IX

Protoporphyrinogen IX

Protoporphyrin IX

Heme

Heme Biosynthetic Pathway of Wolbachia(wBm). Heme is synthesized through a well-defined evolutionarily conserved pathway. Theeukaryotic/prokaryotic/Wolbachia gene namesfor the enzymes involved in each step of thepathway are given in bold.

ALAS, 5-aminolevulinate synthase; ALAD, 5-aminolevulinate dehydratase (also known asPBGS, porphobilinogen synthase,); PBGD,porphobilinogen deaminase; UROS,uroporphyrinogen III synthase; UROD,uroporphyrinogen III decarboxylase; CPO,coproporphyrinogen IX oxidase; PPO,protoporphyrinogen IX oxidase; FC,ferrochelatase.

ALAS/hemA/Wbm0133

ALAD/hemB/Wbm0373

PBGD/hemC/Wbm0777

UROD/hemE/Wbm0001

CPO/hemF/Wbm0709

PPO/hemJ/Wbm0208

FC/hemH/Wbm0719

UROS/hemD/Wbm0728

0.00.20.40.60.81.01.21.4

0 5 20 100Wor

m H

eme

Cont

ent (

nM)

Medium Heme Content (μM)

00.5

11.5

22.5

33.5

0 5 20 100Wor

m H

eme

Cont

ent (

nM)

Medium Heme Content (μM)

1 d2 d3 d4 d5 d6 d

Total worm heme content increases withincreasing medium heme concentration

Total worm heme content increases over time

B. malayi adult worms viability assay

0

1

2

3

4

5

0 1 2 3 4 5 6 7 8 9Days

Viab

ility

Male (3mM SA)Female (3mM SA)

Male (100uM NMMP)Female (100uM NMMP)

Control: Male/Female

B. malayi ex vivo viability assays with heme pathway inhibitors –succinyl acetone & N-methyl mesoporphyrin show Wolbachia hemebiosynthesis is required for worm survival

-Both adult worms & microfilaria are killed by heme pathway inhibitors

(NMMP—FC inhibitor)(SA- ALAD inhibitor)

This suggests Wolbachia heme biosynthesis is criticalfor worm survival. However, heme can also be takenup from the media.

Total RNA

NEBNext® RNA Ultra Library Prep Kit for

Illumina

Sequence-Illumina GAIIX

Map Reads

Assemble TranscriptsFPKMs-Fragments Per Kilobase Mapped reads

Differential Expression Testing

Visualization

Transcriptomics: Using NextGen sequencing technology to examine RNA profiling of Wolbachia

Addition of heme to the media induces “up” and “down” regulation of genes (especially in microfilaria). Interestingly, heme addition down-regulates most Wolbachia heme biosynthesis genes.

Heme homeostasis-B. malayi similar, but not identical to, C. elegans, a free-living nematode

• HRG-1– high affinity transporter– CeHRG-1/BmHRG-1

• 39% identity/60% similar• MRP-5

– heme exporter– CeMRP-5/BmMRP-5

• 48% identity/66% similar• HRG-2

– heme transport into hypodermis– CeHRG-2/BmHRG-2

• 31% identity/51% similar

Summary:--A conundrum exists for understanding the role of heme biosynthesis. Wolbachia genomics, biochemistry and inhibition studies suggest the pathway is

essential for worm survival and expression studies suggest heme regulates Wolbachia heme biosynthesis genes.--Yet Brugia has the functional genes for heme uptake and distribution. In B. malayi and the related nematode D. immitis, expression studies indicate heme

biosynthesis genes are up-regulated in male and female tissues along with Sec and Type IV secretion system components. Genes encoding heme transportand distribution from the extracellular environment are present and functional.

--We hypothesize that Wolbachia helps supply heme for fertility (oogenesis) and perhaps for other requirements for additional heme. One example may befor worm survival in the insect vector host, where exogenous heme may be negligible.

The Search for “Heme Response Genes” (HRGs)

Differential expression: Wolbachia heme biosynthesis genes and genes related to heme binding and utilization are up-regulated, especially in microfilaria.

AM AF L3 mf L4HemAHemBHemCHemDHemEHemFHemJCcmB

Wbm0728Wbm0481

Wbm0719

Wbm0777

μM heme0 5 20 100

Wbm0208Wbm0144

Wbm0014

Heme synthesis and transport appear to be linked to heme binding/utilization in a stage-specific manner.

Ashley N. Luck, Jeremy M. Foster, Silvia Libro, Laurie S. Mazzola, Joanna M. Bybee, Danielle L.Rivizzigno and Barton E. Slatko; Genome Biology Division, New England Biolabs, Ipswich, MA USA

Left panel: Each gene in the “volcano plot” is represented by a dot that is significantly differentially expressed (red) as a function of increasing the heme concentration. Right panel: Wolbachia biosynthesis genes are down-regulated as a function of increasing heme.