Characterization of Brucella abortus mutant strain Δ22915, a potential vaccine candidate

Abstract

Brucellosis, caused by Brucella spp., is an important zoonosis worldwide. Vaccination is an effective strategy for protection against Brucella infection in livestock in developing countries and in wildlife in developed countries. However, current vaccine strains including S19 and RB51 are pathogenic to humans and pregnant animals, limiting their use. In this study, we constructed the Brucella abortus (B. abortus) S2308 mutant strain Δ22915, in which the putative lytic transglycosylase gene BAB_RS22915 was deleted. The biological properties of mutant strain Δ22915 were characterized and protection of mice against virulent S2308 challenge was evaluated. The mutant strain Δ22915 showed reduced survival within RAW264.7 cells and survival in vivo in mice. In addition, the mutant strain Δ22915 failed to escape fusion with lysosomes within host cells, and caused no observable pathological damage. RNA-seq analysis indicated that four genes associated with amino acid/nucleotide transport and metabolism were significantly upregulated in mutant strain Δ22915. Furthermore, inoculation of ∆22915 at 10⁵ colony forming units induced effective host immune responses and long-term protection of BALB/c mice. Therefore, mutant strain ∆22915 could be used as a novel vaccine candidate in the future to protect animals against B. abortus infection.

Figure 1

Characterization of mutant strain ∆22915. A Schematic of BAB_RS22915 gene deletion. A 1036-bp fragment was deleted from the BAB_RS22915 coding sequence. B Transcription of BAB_RS22915 and flanking genes in ∆22915. Transcription of BAB_RS22915 was abolished and had no influence on transcriptional BAB_RS22910 and BAB_RS22920. Data were presented as mean ± SD, and analyzed using a Student’s t test. C Silver staining of bacterial LPS. No difference was seen in LPS patterns between ∆22915 and wild-type strain S2308. Lane Marker: Prestain page ruler (Thermo Scientific, USA); Lane S2308: B. abortus S2308 LPS; Lane ∆22915: ∆22915 LPS. D Bacterial growth curves. Strains ∆22915 and S2308 were cultured in TSB media, and OD₆₀₀ was measured every 4 h to monitor growth. Data were presented as mean ± SD, and analyzed with two-way ANOVA. **P < 0.01; ***P < 0.001.

Figure 2

Intracellular survival and traffic of mutant strain ∆22915 in RAW264.7 cells. A Intracellular survival of ∆22915 was significantly decreased compared to wild-type strain S2308 at 24 and 48 h post infection. Data were presented as mean ± SD, and analyzed with two-way ANOVA. ***P < 0.001. B Determination of LAMP-1-positive BCVs in RAW264.7 cells. LAMP-1-positive BCV ratio was significantly higher for ∆22915-infected cells than S2308-infected cells at 24 h post infection. Data were presented as mean ± SD, and analyzed using a Student’s t test. **P < 0.01. C Representative images of LAMP-1-positive or -negative BCVs of RAW264.7 cells.

Figure 3

Comparison of the persistence of mutant strain ∆22915, wild-type strain S2308 and vaccine strain RB51. Five female BALB/c mice were IP-inoculated with ∆22915, S2308 or RB51 at 1 × 10⁵ CFU/mouse. A Bacterial loads in spleens were determined at 2, 4, 6, 9 and 12 weeks post infection. Persistence of ∆22915 in mice was significantly decreased compared to that of strain S2308 at all time points investigated. Vaccine strain RB51 was cleared at 9 wpi. B There was no significant difference in spleen weights among the uninfected mice, or mice infected with mutant strain ∆22915, or vaccine strain RB51. Spleen weights from S2308-infected mice were significantly increased. Data were presented as mean ± SD (n = 5), and analyzed with two-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Histopathological examination and proinflammatory cytokine determination. A Histological examinations of spleen and kidney at 12 wpi from mice infected with mutant strain ∆22915 and wild-type strain S2308. Non-infected spleens and kidneys were used as normal controls. Strain ∆22915 caused no observable pathological damage in spleens or kidneys. Arrows indicate normal boundaries between red and white pulps in ∆22915-infected spleen, which was similar with uninfected spleen, however, no clear boundary was shown in strain S2308 infected spleen due to tissue damage. Kidney lesions caused by strain S2308 infection are circled. Bars represent 200 μm. B Determination of TNF-α in peripheral blood. TNF-α induction by ∆22915 was significantly lower than by S2308, but higher than by no infection. C Determination of IL-12p40 in peripheral blood. IL-12p40 induction by ∆22915 was significantly lower than by S2308 but higher than by no infection. Data were expressed as mean ± SD (n = 5) and analyzed with two-way ANOVA. ***P < 0.001.

Figure 5

Genetic organization of upregulated gene locus. A BAB_RS17405 encodes a dihydropyrimidinase on chromosome I of B. abortus S2308. It is flanked by a zinc-dependent allantoate amidohydrolase (BAB_RS17410) and a dihydrorhizobitoxine desaturase (BAB_RS17400). B BAB_RS17430 encodes the α-subunit of NADPH dependent glutamate synthase on chromosome I of B. abortus S2308, downstream of β-subunit B of NADPH dependent glutamate synthase (BAB_RS17425). C BAB_RS24460 encodes a substrate-binding protein on chromosome I of B. abortus S2308. It is flanked by genes for two hypothetical proteins without complete coding sequences (BAB_RS24465 and 24470), an ATP-binding protein with ATPase enzymatic activity (BAB_RS24475), two permease proteins (BAB_RS24480 and 24485) and another substrate-binding protein (BAB_RS24455). D BAB_RS30485 encodes a substrate binding protein which is located on chromosome II of B. abortus S2308. It is flanked by two permease proteins (BAB_RS30470 and 30475) and another ATP-binding protein (BAB_RS30480). Both ABC transporters are predicted to be involved in amino acid transport and metabolism. E BAB_RS27765 encodes a putative fumarylacetoacetate (Faa) hydrolase without known function. It is on chromosome II of B. abortus S2308, upstream of a gene for galactose 1-dehydrogenase (BAB_RS27770). F BAB_RS31735 encodes a putative amidohydrolase without known function. It is on chromosome II of B. abortus S2308, downstream of a gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase (BAB_RS31730) gene. Arrows indicate direction of CDS. Except BAB_RS27765 and 31735, all other CDSs are on the complementary strand of the B. abortus S2308 genome. Tags of upregulated genes are indicated by a darker color.

Figure 6

Determination of Brucella antibody and protection by vaccination with mutant strain ∆22915. A Strain ∆22915 induced significantly higher titers of Brucella-specific antibodies than RB51 at all investigated time points. Data were expressed as mean ± SD (n = 5) and analyzed with two-way ANOVA. ***P < 0.001. B Protection efficacy at 12 weeks post vaccination. C Protection efficacy at 16 weeks post vaccination. For protection evaluation, vaccinated mice were challenged with 1 × 10⁴ CFU nalidixic-acid resistant S2308/mouse. One week post challenge, spleens were collected to determine bacterial loads to assess protection efficacy. No bacteria were recovered from the mice of ∆22915-vaccinated group, indicating better protection efficacy than RB51. Data points were individual values of CFU determinations (n = 5) and analyzed using a Student’s t test. **P < 0.01; ***P < 0.001.