MtuA, a lipoprotein receptor antigen from Streptococcus uberis, is responsible for acquisition of manganese during growth in milk and is essential for infection of the lactating bovine mammary gland

Abstract

A mutant strain of Streptococcus uberis (AJS001) that was unable to grow in bovine milk was isolated following random insertional mutagenesis. The level of growth in milk was restored to that of the parental strain (strain 0140J) following addition of MnSO(4) but not following addition of other metal ions. The mutant contained a single insertion within mtuA, a homologue of mtsA and psaA, which encode metal-binding proteins in Streptococcus pyogenes and Streptococcus pneumoniae, respectively. Strain AJS001 was unable to infect any of eight quarters on four dairy cows following intramammary challenge with 10(5) CFU. Bacteria were never recovered directly from milk of these animals but were detected following enrichment in Todd-Hewitt broth in three of eight milk samples obtained within 24 h of challenge. The animals showed no inflammatory response and no signs of mastitis. Three mammary quarters on two different animals simultaneously challenged with 600 CFU of the parental strain, strain 0140J, became colonized, shed high numbers of S. uberis organisms in milk, displayed a marked inflammatory response to infection, and showed overt signs of mastitis. These data indicate that mtuA was required for efficient uptake of Mn(2+) during growth in bovine milk and infection of the lactating bovine mammary gland.

FIG. 1.

Growth of S. uberis in skim milk containing supplements from a chemically defined medium. The data are the geometric means of the numbers of bacteria detected following inoculation of skim milk containing different supplements with 10³ to 10⁴ CFU of either S. uberis 0140J (solid bars) (n = 2) or AJS001 (open bars) (n = 2) and incubation at 37°C for 24 h. The error bars indicate standard errors. Supplements were added from 10× stock solutions, and distilled water was added as a control. Glucose was added to a final concentration of 10 g/liter. The amino acid supplement contained Glu (30 mg/ml); Arg, Trp, Tyr, and His, each at a concentration of 20 mg/ml; and Phe, Met, Ile, Cys, Leu, and Val, each at a concentration of 10 mg/ml. All the amino acids were in the l configuration. The trace element supplement contained MgSO₄, (2 g/liter), FeSO₄ (100 mg/liter), and MnSO₄ (100 mg/liter). The vitamin supplement contained riboflavin (4 mg/liter), thiamine (4 mg/liter), folate (1 mg/liter), p-aminobenzoate (1 mg/liter), pantothenate (8 mg/liter), pyridoxamine (8 mg/liter), biotin (0.1 mg/liter), and niacinamide (20 mg/liter).

FIG. 2.

Growth of S. uberis in skim milk containing trace metals. The data are the geometric means of the number of S. uberis 0140J CFU (solid bars) (n = 2) or AJS001 CFU (open bars) (n = 2) detected in skim milk following incubation at 37°C for 24 h. The error bars indicate standard errors. The mean number of bacteria at zero time is shown (T = 0). Each metal ion was added at a concentration of 4 μM as the sulfate salt, and distilled water was added as a diluent control.

FIG. 3.

Growth of S. uberis in skim milk containing different concentrations of Mn²⁺. The data are the geometric means of the number of S. uberis 0140J CFU (solid bars) (n = 3) or AJS001 CFU (open bars) (n = 5) detected in skim milk containing MnSO₄ at different concentrations following incubation at 37°C for 24 h. The error bars indicate standard errors. Both strains were inoculated into milk to a density of ∼10³ CFU/ml. S. uberis strain 0140J was investigated only in milk samples containing 0 and 4 μM MnSO₄.

FIG. 4.

mtu ABC transporter operon of S. uberis and other LraI loci. The open reading frames whose designations end withA encode solute-binding lipoproteins; the open reading frames whose designations end with B encode ATP-binding proteins; and the open reading frames whose designations end with C encode hydrophobic membrane proteins. psaD encodes a peroxidase not encoded at comparable S. uberis or S. pyogenes loci. Expression of the mtu operon is thought to be coordinated by a metallorepressor protein homologue (Regl.) located immediately upstream of mtuA. The open arrows indicate directions of transcription, and putative hairpin loops are shown between open reading frames. The location and orientation of ISS1 in S. uberis AJS001 are also shown.

FIG. 5.

SDS-PAGE and Western blot analyses of whole-cell lysates of S. uberis, showing detection of MtuA. Western blotting with antiserum raised against the CAL-n-FLAG-′MtuA fusion protein (A) and SDS-PAGE (B) of whole-cell lysates from S. uberis revealed a single reactive protein band at around 37 kDa (arrow) in strain 0140J (lane 2 in panel A) and no cross-reactive proteins in strain AJS001 (lane 3 in panel A) resulting from matched sample loadings (lanes 2 and 3 in panel B). Lane 1 contained molecular weight markers, and the mass of each marker protein (in kilodaltons) is indicated on the left.

FIG. 6.

Inflammatory response and bacterial recovery following experimental challenge of the bovine mammary gland with S. uberis. The data are the geometric means of the number of somatic cells (A) or bacteria (B) detected in milk samples obtained before (milking 0) and after (milkings 1 to 7) challenge with S. uberis 0140J (solid bars) (n = 3) or AJS001 (open bars) (n = 8). Following challenge with strain AJS001, bacteria were detected in three of eight challenged quarters on two animals following enrichment culture (asterisks).