The entomopathogenic fungus Metarhizium anisopliae alters ambient pH, allowing extracellular protease production and activity

Ambient ph regulates the expression of virulence genes of Metarhizium aniso pliae, but it was unknown if M. anisopliae can regulate ambient pH. Mutants of M. anisopliae altered in production of oxalic acid were evaluated for t he interrelationship of ambient pH, buffering capacity added to media, grow th, and generation of extracellular proteases and ammonia. Wild-type and ac id-overproducing mutants [Acid(+)] grew almost as well at ph 8 as at ph 6, but acid-non-producing [Acid(-)] mutants showed limited growth at ph 8, ind icating that acid production is linked to the ability to grow at higher ph. Production of ammonia by M. anisopliae was strongly stimulated by low leve ls of amino acids in the medium when cells were derepressed for nitrogen an d carbon. Likewise, although Aspergillus fumigatus and Neurospora crassa pr oduced some ammonia in minimal media, addition of low levels of amino acids enhanced production. Ammonia production by A. fumigatus, N. crassa and M. anisopliae increased the ph of the medium and allowed production of subtili sin proteases, whose activities are observed only at basic ph. In contrast, protease production by the Acid(+) mutants of M. anisopliae was greatly re duced because of the acidification of the medium. This suggests that alkali nization by ammonia production is adaptive by facilitating the utilization of proteinaceous nutrients. collectively, the data imply that ammonia may h ave functions related to regulation of the microenvironment and that it rep resents a previously unconsidered virulence factor in diverse fungi with th e potential to harm tissues and disturb the host's immune system.