Additional file 1: Table S1. of Rapidly-growing mycobacterial infection: a recognized cause of early-onset prosthetic joint infection

(Clinical features and outcomes of 11 non-tuberculous mycobacterial and 5 tuberculous prosthetic joint infections). (DOC 67 kb

Yeast two hybrid analysis of Cna1 and Crz1 interaction.

<p>BD and AD designate GAL4 binding and activation domains fused to Cna1 and Crz1 respectively, where applicable. Vector equals empty vector. All combinations of transformants grew in the absence of Leu and Trp confirming plasmid retention in the parent strain. However, only yeast transformed with plasmids containing <i>CNA1</i> and <i>CRZ1</i> grew in the absence of Ade confirming an interaction which allows expression of Ade biosynthetic machinery. Quantitative analysis of β-galactosidase activity was performed using ONPG as a substrate. For this test, the cells were grown on SD Leu^-Trp^- plates with or without 1 µg/ml FK506. Error bars represent standard deviation.</p

Calcineurin protein is required for targeting of Crz1-GFP to the nuclei, but not to the cytosolic puncta.

<p>The strain used in all panels is Crz1-GFP:<i>Δcna1.</i> (A) Crz1-GFP is excluded from the nuclei in <i>Δcna1</i> grown at room temperature; (B) 1 M NaCl triggers punctate Crz1-GFP localization even in the absence of calcineurin; (C) addition of calcium fails to trigger nuclear translocation of Crz1-GFP in <i>Δcna1</i>.</p

The Crz1/Sp1 Transcription Factor of <em>Cryptococcus neoformans</em> Is Activated by Calcineurin and Regulates Cell Wall Integrity

<div><p><em>Cryptococcus neoformans</em> survives host temperature and regulates cell wall integrity via a calcium-dependent phosphatase, calcineurin. However, downstream effectors of <em>C. neoformans</em> calcineurin are largely unknown. In <em>S. cerevisiae</em> and other fungal species, a calcineurin-dependent transcription factor Crz1, translocates to nuclei upon activation and triggers expression of target genes. We now show that the <em>C. neoformans</em> Crz1 ortholog (Crz1/Sp1), previously identified as a protein kinase C target during starvation, is a <em>bona fide</em> target of calcineurin under non-starvation conditions, during cell wall stress and growth at high temperature. Both the calcineurin-defective mutant, Δ<em>cna1,</em> and a <em>CRZ1/SP1</em> mutant (Δ<em>crz1</em>) were susceptible to cell wall perturbing agents. Furthermore, expression of the chitin synthase encoding gene, <em>CHS6,</em> was reduced in both mutants. We tracked the subcellular localization of Crz1-GFP in WT <em>C. neoformans</em> and <em>Δcna1</em> in response to different stimuli, in the presence and absence of the calcineurin inhibitor, FK506. Exposure to elevated temperature (30–37°C vs 25°C) and extracellular calcium caused calcineurin-dependent nuclear accumulation of Crz1-GFP. Unexpectedly, 1M salt and heat shock triggered calcineurin-independent Crz1-GFP sequestration within cytosolic and nuclear puncta. To our knowledge, punctate cytosolic distribution, as opposed to nuclear targeting, is a unique feature of <em>C. neoformans</em> Crz1. We conclude that Crz1 is selectively activated by calcium/calcineurin-dependent and independent signals depending on the environmental conditions.</p> </div

Sequence analysis of <i>C. neoformans</i> Crz1 and comparison with other fungal species.

<p>(A) Diagram of the Crz1 functional domains. (B) Phylogenetic tree of fungal CRZ proteins based on the alignment of their Zn finger DNA-binding domains. Bootstrap values are indicated. <i>S. cerevisiae</i> Swi5 and Ace2 represent transcription factors related to ScCrz1 Cn_H99, <i>Cryptococcus neoformans</i> serotype A; Cn_JEC21, XP_566613, <i>C. neoformans</i> serotype D; An, BAE94327, <i>Aspergillus nidulans</i>; Ao, BAE57003, <i>Aspergillus oryzae</i>; Af, EAL88401, <i>Aspergillus fumigatus</i>; Mo, XP_359644, <i>Magnaporthe oryzae</i>; Nc, EAA32849, <i>Neurospora crassa</i>; EAQ88414, Chg, <i>Chaetomium globosum</i>; Gz, XP_381517, <i>Gibberella zeae</i>; Ci, XP_001244584, <i>Coccidioides immitis</i>; Pn, EAT87393, <i>Phaeosphaeria nodorum</i>; Dh, CAG84727, <i>Debaryomyces hansenii</i>; Td, AAZ04388, <i>Torulaspora delbrueckii</i>; Ag, AAS51722, <i>Ashbya gossypii</i>; Sc, CAA95889, <i>Saccharomyces cerevisiae</i>; Kl, CAG99429, <i>Kluyveromyces lactis</i>; Yl, CAG80473, <i>Yarrowia lipolytica</i>; Ca, EAK97605, <i>Candida albicans</i>; Cgl, CAG62620, <i>Candida glabrata</i>; Sp, Q09838, <i>Schizosaccharomyces pombe</i>; Cd, CAX43071, <i>Candida dubliniensis</i>; Sc_Ace2, CAA97702, <i>Saccharomyces cerevisiae</i>; Sc_Swi5, CAA90369, <i>Saccharomyces cerevisiae</i>. Indicate what the red numbers mean.</p

<i>Δcna1</i> and <i>Δcrz1</i> mutants share sensitivity to cell wall perturbing agents.

<p>WT H99, <i>Δcna1, Δcrz1 and Δcrz1-rec</i> (serially-diluted 10-fold, 10⁶–10 cells/spot from left to right) were spotted on YPD plates supplemented with NaCl, CaCl₂, calcofluor white (CFW) or Congo Red as indicated, and incubated at 30°C. Thermosensitivity of the mutants was tested at 37°C on YPD plates.</p

FK506 causes exclusion of heat shock-induced Crz1-GFP puncta from the nucleus.

<p><i>C. neoformans</i> cells growing at room temperature (A) were treated with 10 µg/ml FK506 for 1 hour (B) and then exposed to 42°C for 5 min. The fluorescence pattern was compared in FK506-treated and untreated cells within 45 minutes following heat shock (C, D).</p

Salt stress and high temperature trigger punctate localization of Crz1-GFP.

<p>(A) Cells grown at room temperature show predominantly cytosolic and occasional nuclear Crz1-GFP localization, while Crz1-GFP is sequestered in cytosolic puncta in cells treated with 1 M NaCl (B). In cells exposed to 42°C, puncta are localized in and around nuclei (C).</p