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Aspergillus flavus is one of the predominant causative organisms of mycotic keratitis in tropical parts of the world. Extracellular proteins are the earliest proteins that come in contact with the host and have a role in the infection process. Exoproteins of A. flavus isolated from infected cornea, sputum and a saprophyte were pooled and identified using high resolution mass spectrometry in order to get the total exoproteome from cultures isolated from different sources. A total of 637 proteins was identified from the pooled A. flavus exoproteome. Analysis based on GO annotations of the 637 identified proteins revealed that hydrolases form the predominant class of proteins in the exoproteome. Interestingly, a greater proportion of the exoproteins seem to be secreted through the non-classical pathways. This data represent the first in-depth analysis of the representative A. flavus exoproteome of a large set of isolates from distinct sources. This data have been deposited to the ProteomeXchange with identifier PXD001296.

Aspergillus flavus infects the human eye leading to keratitis. Extracellular proteins, the earliest proteins that come in contact with the host and virulence related exoproteins, were identified in the fungus isolated from infected cornea. Virulence of the corneal isolates was tested in the Galleria mellonella larvae model and those isolates showing higher virulence were taken for subsequent exoproteome analysis. High resolution two-dimensional electrophoresis and mass spectrometry were used to generate A. flavus exoproteome reference map as well as to profile most of the exoproteins. Analysis of the identified proteins clearly shows the major biological processes that they are involved in. Nearly 50% of the exoproteins possess catalytic activity and one of these, an alkaline serine protease (Alp1) is present in high abundance as well as multiple proteoforms. Many proteins in the A. flavus exoproteome have been shown to be virulence factors in other pathogens indicating the probable role for these proteins in the corneal infection as well. Interestingly, the majority of the exoproteins do not have secretory signal indicating that they are secreted through the non-classical pathway. Thus, this study provides a clue to the early strategies employed by the pathogen to establish an infection in an immunocompetent host. Biological significance The outcome of a fungal infection in an immunocompetent human eye depends on the ability of the fungus to overcome the host defense and propagate itself. In this process, the earliest events with respect to the fungal proteins involved include the secretory proteins of the invading organism. As a first step towards understanding the role of the extracellular proteins, exoproteome profile of the fungal isolates was generated. The fungal isolates from cornea showed a distinct pattern of the exoproteome when compared to the saprophyte. Since corneal isolates also showed higher virulence in the insect larval model, presumably the proteins elaborated by the corneal isolates are virulence related. One of the abundant proteins is an alkaline serine protease and this protein exists as multiple proteoforms. This study reports the comprehensive profile of exoproteome and reveals proteins that are potential virulence factors.

The aim of this study is to examine the in vivo role of a small heat-shock protein (sHsp18) from Mycobacterium leprae in the survival of heterologous recombinant hosts carrying the gene encoding this protein under different environmental conditions that are normally encountered by M. leprae during its infection of the human host. Using an Escherichia coli system where shsp18 expression is controlled by its native promoter, we show that expression of shsp18 is induced under low oxygen tension, nutrient depletion and oxidative stress, all of which reflect the natural internal environment of the granulomas where the pathogen resides for long periods. We demonstrate the in vivo chaperone activity of sHsp18 through its ability to confer survival advantage to recombinant E. coli at heat-shock temperatures. Additional evidence for the protective role of sHsp18 was obtained when Mycobacterium smegmatis harbouring a copy of shsp18 was found to multiply better in human macrophages. Furthermore, the autokinase activity of sHsp18 protein demonstrated for what is believed to be the first time in this study implies that some of the functions of sHsp18 might be controlled by the phosphorylation state of this protein. Results from this study suggest that shsp18 might be one of the factors that facilitate the survival and persistence of M. leprae under stress and autophosphorylation of sHsp18 protein could be a mechanism used by this protein to sense changes in the external environment.

BackgroundSmall heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen.M. leprae, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloningM. lepraegenes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen ofM. lepraeis scanty.ResultsThe gene encodingMycobacterium lepraesmall heat shock protein (sHsp18) was amplified from biopsy material of leprosy patients, and cloned and expressed inE. coli. The localization andin vitrocharacterization of the protein are detailed in this report. Data show that major portion of the protein is localized in the outer membrane ofE. coli. The purified sHsp18 functions as an efficient chaperone as shown by their ability to prevent thermal inactivation of restriction enzymesSmaI andNdeI. Physical interaction of the chaperone with target protein is also demonstrated. Size exclusion chromatography of purified protein shows that the protein can form multimeric complexes underin vitroconditions as is demonstrated for several small heat shock proteins.ConclusionThe small heat shock protein sHsp18 ofM. lepraeis a chaperone and shows several properties associated with other small heat shock proteins. Membrane association andin vitrochaperone function of sHsp18 shows that the protein may play a role in the virulence and survival ofM. lepraein infected host.