Roles of His-rich hpn and hpn-like proteins in Helicobacter pylori nickel physiology

Abstract

Individual gene-targeted hpn and hpn-like mutants and a mutant with mutations in both hpn genes were more sensitive to nickel, cobalt, and cadmium toxicity than was the parent strain, with the hpn-like strain showing the most metal sensitivity of the two individual His-rich protein mutants. The mutant strains contained up to eightfold more urease activity than the parent under nickel-deficient conditions, and the parent strain was able to achieve mutant strain activity levels by nickel supplementation. The mutants contained 3- to 4-fold more and the double mutant about 10-fold more Ni associated with their total urease pools, even though all of the strains expressed similar levels of total urease protein. Hydrogenase activities in the mutants were like those in the parent strain; thus, hydrogenase is fully activated under nickel-deficient conditions. The histidine-rich proteins appear to compete with the Ni-dependent urease maturation machinery under low-nickel conditions. Upon lowering the pH of the growth medium from 7.3 to 5, the wild-type urease activity increased threefold, but the activity in the three mutant strains was relatively unaffected. This pH effect was attributed to a nickel storage role for the His-rich proteins. Under low-nickel conditions, the addition of a nickel chelator did not significantly affect the urease activity of the wild type but decreased the activity of all of the mutants, supporting a role for the His-rich proteins as Ni reservoirs. These nickel reservoirs significantly impact the active urease activities achieved. The His-rich proteins play dual roles, as Ni storage and as metal detoxification proteins, depending on the exogenous nickel levels.

FIG. 1.

Viability loss due to metal toxicity. Strains: H. pylori 43504 (♦), hpn (▪), hpn-like (▴), and the double-mutant strain (×). (A) Survival in PBS with no added metals. The experiment was performed in triplicate, and the data shown are means from all three data sets for each strain. No significant differences among strains were observed. (B) Survival in the presence of 1 mM NiCl_2. The data are from four independent experiments, each one sampled three times; thus, each data point is the mean of 12 readings. The standard deviation ranged between 3 to 20% of the mean values; all hpn mutant strain results are significantly different (P < 0.05) from the wild type at 6 h and all later time points, while the other two mutant strains are significantly different from the wild type (P < 0.05) at 4 h and all later plating points. (C) Survival in the presence of 10 μM CdCl_2. The data are from two independent experiments, each one sampled twice; thus, each point is the mean of four values. The values for the hpn-like mutant and the double mutant are significantly different (P < 0.05) from the wild type at 6 h and later, whereas the hpn mutant is different at the 9- and 12-h time points (P < 0.05). (D) Survival in the presence of 20 μM CoCl_2. The data are from two independent experiments, each one sampled twice; thus, each point is the mean of four values. The His-rich protein mutants are significantly less tolerant than the wild type at the 9- and 11-h points (P < 0.05), and the double mutant is significantly less tolerant than the wild type at 6 h and later sampling points.

FIG. 2.

Urease activities and protein as a function of the nickel content of the medium. (A) Urease activities of cells grown without nickel supplementation on BA plates. The results shown are means and standard deviations of six replicate samples (these six represent three measurements from each of two independent cultures); the experiment was performed four additional times, with similar results. Among the five experiments, the double-mutant strain mean exceeded the Hpn strain in all cases, by a total range of 5 to 65%. All three mutant strains have significantly greater urease activities than do the wild type (P < 0.01), based on Student t test analysis. (B) Urease activities of cells grown with (1 or 5 μM NiCl₂) or without nickel supplementation. Cells were grown on BA plates. The strains are as follows: wild-type H. pylori (black), the hpn strain (white), the hpn-like strain (striped), and the double-mutant strain (gray). The measurements were performed in triplicate (one culture assayed three times); the wild-type urease activities are greater under Ni supplementation conditions than without Ni supplementation (P < 0.01), based on Student t test analysis, and the mutant strain activities exceed the wild-type activity under zero nickel conditions (P < 0.05). The entire experiment was done two other times, with results similar to those shown. (C) Anti-UreA immunoblots performed on gel-resolved peptides of crude cell extracts from cells grown in non-Ni-supplemented medium. Crude extracts were loaded (5 μg protein) with the double-mutant (lane 1), hpn-like (lane 2), hpn (lane 3), or wild type (lane 4) strain. Lane M is the low-range prestained molecular mass marker, with sizes indicated on the right. Densitometric scanning of each lane revealed no significant differences in (UreA) protein amounts between strains.

FIG. 3.

Effects of pH and dimethyl glyoxime on urease activities. Urease activities of strains grown in BHI medium with 5% fetal bovine serum at pH 7.3 (white) or pH 5 (black) or with the nickel chelator dimethyl glyoxime at pH 7.3 (gray) are shown. The results shown are means and standard deviations of six replicate samples; these six replicates are from two independent cultures, each sampled three times. The entire experiment was performed two additional times (again with two cultures for each experiment), with results similar to those shown. At pH 5, the urease activities of the wild type (WT) are significantly higher (P < 0.05) than at pH 7.3, and the chelator-added condition was not different from that without chelator (pH 7.3). The activities of the chelator-added condition for the mutants are significantly lower than without chelator (P < 0.01).