Abstract
Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.
Highlights
Probiotics are defined as live microorganisms which, when administered in sufficient amounts, confer health benefits in the host in addition to their nutritional effects [1]
To identify genes involved in the augmentation of host innate immunity by L. casei, a total number of 9408 Tn5 insertion mutants from the mutant library were screened for their J1 phage sensitivity using a 96-well plate assay
Inverse PCR and sequencing of the DNA regions flanking the transposon insertion sites of each J1 phage-resistant mutant indicated that asnH, dnaJ, and dnaK were disrupted by insertion of the transposon element: among the 9 Tn5 mutants, 3 mutants (MT77, MT4156, and MT8105) harbored Tn5 insertions in asnH, 5 mutants (MT2890, MT3332, MT5740, MT6674, and MT7398)
Summary
Probiotics are defined as live microorganisms which, when administered in sufficient amounts, confer health benefits in the host in addition to their nutritional effects [1]. L. casei strain Shirota has been widely studied for its probiotic effects including immunomodulating activity It enhances NK cell activity by dairy intake [3,4] and prevents and reduces the risk of recurrence of bladder cancer by oral administration in human [5,6]. This strain enhances anti-infectious activity against Listeria monocytogenes [7,8] and Pseudomonas aeruginosa [9] and inhibits the growth of transplantable allogeneic and syngeneic mouse tumors in several experimental animal models [10,11,12,13,14]. The detailed molecular mechanisms involved in these processes have not been understood
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