Investigating the importance of selected surface-exposed loops in HpuB for hemoglobin binding and utilization by Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae is the etiological agent of the sexually transmitted infection gonorrhea. The pathogen is a global health challenge since no protective immunity results from infection, and far fewer treatment options are available with increasing antimicrobial resistance. With no efficacious vaccines, researchers are exploring new targets for vaccine development and innovative therapeutics. The outer membrane TonB-dependent transporters (TdTs) produced by N. gonorrhoeae are considered promising vaccine antigens as they are highly conserved and play crucial roles in overcoming nutritional immunity. One of these TdTs is part of the hemoglobin transport system comprised of HpuA and HpuB. This system allows N. gonorrhoeae to acquire iron from hemoglobin (hHb). In the current study, mutations in the hpuB gene were generated to better understand the structure-function relationships in HpuB. This study is one of the first to demonstrate that N. gonorrhoeae can bind to and utilize hemoglobin produced by animals other than humans. This study also determined that when HpuA is absent, mutations targeting extracellular loop 7 of HpuB led to defective hHb binding and utilization. However, when the lipoprotein HpuA is present, these loop 7 mutants recovered their ability to bind hHb, although the growth phenotype remained significantly impaired. Interestingly, loop 7 contains putative heme-binding motifs and a hypothetical α-helical region, both of which may be important for the use of hHb. Taken together, these results highlight the importance of loop 7 in the functionality of HpuB in binding hHb and extracting and internalizing iron.

Keywords: N. gonorrhoeae; N. meningitidis; TonB-dependent transporter; hemoglobin; hemoglobin–haptoglobin receptor; lipoprotein.

Fig 1

Hypothetical topology map of HpuB as a foundation for mutagenesis. The putative loops are extracellularly located with loop helices represented by coil-like regions. The loop number is shown above each loop. Beta strands are shown in green within the outer membrane. The orange arrows indicate the locations of the deletion mutations generated in this study. Image created with BioRender.com.

Fig 2

Diagram demonstrating how hpuB mutations were moved into the gonococcal chromosome generating gonococcal strains that were either HpuA− (A) or HpuA+ (B). Wild-type and mutated versions of hpuB were cloned into a complementation vector, behind an IPTG-inducible promoter (lacPOPO) and ribosome-binding site. Linearized plasmids* (Table 1) containing either the wild-type (pGSU442) or mutated versions of hpuB* (pGSU443-449) were used to transform piliated recipient N. gonorrhoeae, resulting in the insertion into an ectopic site between the aspC and lctC loci. (A) The recipient for transformation was RSC150, which contains an Ω insertion in the upstream hpuA gene, resulting in a transformant strain that is hpuA− and hpuB− in the native locus, due to the polar nature of the transposon. Transformation resulted in strains with wild-type hpuB (RSC276) in the ectopic site or mutated versions of hpuB* (RSC277-283) in the ectopic site. (B) The recipient for transformation was RSC275, which contains an Ω insertion in the downstream hpuB gene, resulting in a transformant strain that is hpuA+ hpuB− in the native locus. Transformation resulted in strains with wild-type hpuB (RSC284) in the ectopic site or mutated versions of hpuB* (RSC285-291) in the ectopic site. The symbol 5′ to the hpuA gene indicates the promoter that drives the expression of the hpuAB operon, which is repressed by iron (Fe).

Fig 3

Mutated hpuB genes were created and confirmed to be expressed from an inducible promoter in an ectopic site in the gonococcal chromosome. HpuB mutant strains were grown on GC medium base (GCB) plates containing DFO (Fe limiting), with (+) or without (−) 1 mM IPTG. Cells were collected from plates and resuspended into phosphate-buffered saline (PBS). From these suspensions, whole-cell lysates were prepared by standardizing the cell suspensions to an OD₆₀₀ of 1, pelleting cells, and resuspending pellets in lysis buffer. Western blots were performed to characterize the production of HpuB in the presence or absence of IPTG using an anti-HpuB antibody. The strain with the WT hpuB gene in the ectopic site and native hpuA gene was used as a positive control (A+B+). The strain lacking both native hpuA and hpuB genes (A−B−) was used as a negative control. Data shown are representative of three biological replicates.

Fig 4

Loop 7 HpuB mutants are impaired for growth on hemoglobin as a sole iron source. Gonococci were grown on GCB/DFO/IPTG plates before being resuspended in Chelex (Bio-Rad)-treated defined medium (CDM). The cell suspensions were standardized to an OD₆₀₀ of 0.002 before being added to a 96-well plate containing DFO, IPTG, and 1 µM hHb. Cells were grown for 21 hours, during which the OD₆₀₀ was recorded in 30-minute intervals to assess the growth of strains in the mutant hpuA background (A) and the wild-type hpuA background (B). A−B− is used as a negative control and A+B+ as a positive control. A+B− as well as A−B+ also represent controls as both proteins are thought to be required for growth. From the growth curves, the AUC was calculated using GraphPad Prism then normalized to growth by the A+B+ strain. The data from three biological replicates were analyzed to generate the means and standard deviations shown. Statistically significant differences were identified by using Student’s t-test relative to the A+B+ strain (^*P < 0.05; ^**P < 0.005) and the A−B− strain (^#P < 0.05; ^##P < 0.005).

Fig 5

Loop 7 HpuB mutants recover hHb binding ability when HpuA is produced. Gonococcal HpuB mutants were grown on GCB/DFO/IPTG plates before being resuspended in PBS to an OD₆₀₀ of 1. Cell suspensions were added to a 96-well ELISA plate and allowed to dry prior to blocking. The ELISA plate was then probed with horseradish peroxidase (HRP)-conjugated hHb before being washed and developed with a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The binding ability of strains without (A) and with (B) native hpuA was assessed. The absorbance at 450 nm was read to quantify the signal. A−B− is used as a negative control and A+B+ as a positive control. A+B− and A−B+ are also used as controls since both HpuA and HpuB are thought to be required for binding. All the strains were normalized to A+B+ and showed as a percentage of A+B+. Three biological replicates are represented with their means and standard deviation shown. Student’s t-test was used to assess the statistically significant differences relative to A+B+ (^*P < 0.005; ^**P < 0.0005).

Fig 6

Mutated hpuB variants are expressed on the gonococcal cell surface and susceptible to trypsin digestion. Strains were grown on GCB/DFO/IPTG plates and resuspended in PBS to an OD₆₀₀ of 0.4. Iron-starved whole cells were then treated with trypsin for 0, 10, 20, 30, and 40 minutes before the reaction was stopped with aprotinin. NT represents no treatment. Next, the cells were pelleted, and the lysates were subjected to SDS-PAGE and western blot. The blots were probed with anti-HpuB antibody followed by an AP-conjugated IgG secondary antibody. Nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolylphosphate (BCIP) was used to develop the blots. (A) A+B+ shows the positive control trypsin digestion pattern. (B) The digestion pattern of the mutants. The black arrows indicate the proteolytic products seen in the positive control.

Fig 7

The deletions in loop 7 moved histidine 548 further away from the closest heme group. HpuA-HpuB-Hb structure. AlphaFold2 was used to obtain predicted models of the HpuA-HpuB-Hb complexes. The AlphaFold2 models were generated with wild-type HpuB (A) or with the indicated mutations in HpuB (B, C, and D). A known Hb-heme crystal structure (PDB:1hho) was super-imposed on these models to illustrate the position of heme groups. HpuB is in green with H548 highlighted in red, HpuA in blue, Hb in red, and heme groups in white with Fe²⁺ as a red dot. The distance in angstroms (Å) between the heme group and histidine at position 548 is indicated.

Fig 8

N. gonorrhoeae can bind hemoglobin produced by animals other than humans. A competition assay was used to establish whether N. gonorrhoeae can bind to Hb from mouse (mHb), pig (pHb), and rat (rHb). A+B+ was grown on GCB/DFO/IPTG plates before being resuspended in PBS to an OD₆₀₀ of 1. The cell suspension was added to a 96-well ELISA plate and allowed to dry prior to blocking. The ELISA plate was then probed with either no Hb (i.e., blocker), 5 nm hHb-HRP without competitor, or 5 nm hHb-HRP + 20× (100 nm) excess competitor. The competitors used were unlabeled hHb, mHb, pHb, or rHb. The next step was to develop with TMB. 5 nm hHb-HRP + no competitor represents the positive control, and the blocker condition represents the negative control. Statistically significant differences were assessed by Student’s t-test relative to the no competitor condition (^*P < 0.0005) and the blocker condition (^#P < 0.0005). The mean and standard deviation of four biological replicates are shown.

Fig 9

N. gonorrhoeae can grow on hemoglobins produced by species other than human, as a sole iron source. A growth assay was used to establish whether N. gonorrhoeae could grow on different Hb species, as their sole iron source. A+B+ was grown on GCB/DFO/ IPTG plates before being resuspended into CDM. The cell suspension was standardized to an OD₆₀₀ of 0.002 before being added to a 96-well plate containing DFO and IPTG with no Hb or 1 µM human, mouse, pig, or rat Hb. Cells were grown for 21 hours while the OD₆₀₀ was recorded in 30-minute intervals to assess the growth of A+B+ with hemoglobin of different species. The human Hb condition represents the positive control, and the no Hb condition represents the negative control. From the growth curves, the AUC was calculated using GraphPad Prism then normalized to growth on human Hb. Three biological replicates are represented with their means and standard deviation shown. Statistically significant differences were assessed by Student’s t-test relative to the human Hb condition (^*P < 0.05) and no Hb (^#P < 0.05).