Production and Quantification of Virulence Factors in Malassezia Species

Inès Hadrich; Nahed Khemekhem; Sourour Neji; Houaida Trablesi; Amin Ilahi; Hayet Sellami; Fattouma Makni; Ali Ayadi

doi:10.33073/pjm-2022-047

Figures & Tables

Different Malassezia species biofilm quantification after crystal violet staining (Abs CV). Error bars represent a standard deviation.

Malassezia species colonies of A) high lipase activity, B) very high lipase activity, C) high phospholipase activity, D) very high phospholipase activity, E) keratinase activity null, F) very high keratinase activity.

The sequences of primers and probes used in RT-qPCR for gene expression studies in 20 Malassezia globosa isolates_

Enzymes	Names	Sequences
Keratinase	KerF	5’-ACGTCATGCTCAGATTGCAG-3’
	KerR	5’-GACTTCCGCGAAGAACAAAG-3’
	KerP	5’-FAM-CACAATTGCTCCCGATACCT-MGB
Lipase	LipF	5’-ACCCAACATTTGCTTCGTTC-3’
	LipR	5’-TCAATTATCAATGGTCGCGA-3’
	LipP	5’-FAM-CCTTGGTGGTCGTCAAAGTT-MGB
Phospholipase	PhosF	5’-AACTGGTGGATTTGCTGACC-3’
	PhosR	5’-CTTTACGGGTCCAAGGTGAA-3’
	PhosP	5’-FAM-CCGTGTGCCATTCTACATTG-MGB
Actin	ActF	5’-CTCTCCTTGTACGCCTCTGG-3’
	ActR	5’-TTGACAAGATGCTCCGTCAG-3’
	ActP	5’-TET-GTATTGTGCTGGACTCGGGT-MGB

Level of phospholipase, lipase, and keratinase production by Malassezia species_

	Phospholipase activity number of isolates (rate of isolates)				Lipase activity number of isolates (rate of isolates)			Keratinase activity number of isolates (rate of isolates)
	Mean (Pz) ±SD	very high n (%)	high n (%)	Nul n (%)	Mean (Pz) ± SD	very high n (%)	high n (%)	Mean (Pz) ± SD	very high n (%)	high n (%)	null
Malassezia globosa (N = 20 )	0.64 ±0.03	9(45)	11 (55)	0(0)	0.55±0.02	19 (95)	1(5)	0.65±0.02	10 (50)	6 (30)	4(20)
Malassezia restricta (N = 10)	0.76 ±0.02	0(0)	7(70)	3 (30)	0.36 ±0.03	10 (100)	0(0)	0.69 ±0.02	0(0)	2 (20)	8 (80)
Malassezia furfur (N = 14)	0.64 ±0.02	2 (14.29)	12 (85.71)	0(0)	0.45 ±0.03	14 (100)	0(0)	0.67±0.02	1(7.15)	6 (42.85)	7(50)
Malassezia sympodialis (N = 7)	0.67 ±0.02	1 (14.29)	6 (85.71)	0(0)	0.54 ±0.02	5 (71.43)	2 (28.57)	0.62 ±0.03	1 (14.3)	3 (42.85)	3 (42.85)
Malassezia slooffiae (N = 5)	0.64 ±0.02	2(40)	3 (60)	0(0)	0.4 ±0.04	5(100)	0(0)	0.67±0.02	2(40)	3 (60)	0(0)
Malassezia pachydermatis (N = 21)	0.64 ±0.03	9 (42.86)	12 (57.14)	0(0)	0.31 ±0.02	21 (100)	0(0)	0.68±0.02	3 (14.29)	8 (38.5)	10 (47.66)
Total	0.66 ±0.02	23 (29.87)	51 (66.23)	3(3.9)	0.48 ±0.02	74 (91.1)	3 (3.9)	0.66 ±0.02	17(22.08)	28 (36.36)	32 (41.56)

Enzymatic activities of Malassezia species isolated from different clinical sites_

		Folliculitis (N = 12)	Neonates group (N = 20)	Pityriasis versicolor (N = 10)	Control group (N = 35)
Phospholipase activity Mean (Pz) ± SD	Malassezia globosa (N = 20)	0.69 ± 0.01 (5/12)	– (0/20)	0.74 ± 0.02 (8/10)	0.73 ± 0.02 (7/35)
	Malassezia restrict (N = 10)	0.67 ± 0.01 (1/12)	– (0/20)	0.8 ± 0.02 (1/10)	0.83 ± 0.02 (8/35)
	Malassezia furfur (N = 14) Malassezia sympodialis (N = 7)	0.74 ± 0.01 (6/12) – (0/12)	0.70 ± 0.02 (1/20) – (0/20)	0.69 ± 0.02 (1/10) – (0/10)	0.71 ± 0.01 (6/35) 0.78 ± 0.02 (7/35)
	Malassezia slooffiae (N = 5)	0 (0/12)	– (0/20)	– (0/10)	0.77 ± 0.02 (5/35)
	Malassezia pachydermatis(N = 21)	0 (0/12)	0.66 ± 0.02 (19/20)	– (0/10)	0.79 ± 0.01 (2/35)
	Total	0.70 ± 0.01	0.68 ± 0.02	0.74 ± 0.02	0.77 ± 0.02
Lipase activity Mean (Pz) ± SD	Malassezia globosa (N = 20)	0.36 ± 0.01 (5/12)	– (0/20)	0.36 ± 0.02 (8/10)	0.38 ± 0.02 (7/35)
	Malassezia restricta (N = 10)	0.37 ± 0.01 (1/12)	– (0/20)	0.32 ± 0.02 (1/10)	0.36 ± 0.02 (8/35)
	Malassezia furfur (N = 14) Malassezia sympodialis (N = 7)	0.32 ± 0.02 (6/12) – (0/12)	0.39 ± 0.02 (1/20) – (0/20)	0.28 ± 0.01 (1/10) – (0/10)	0.36 ± 0.02 (6/35) 0.48 ± 0.02 (7/35)
	Malassezia slooffiae (N = 5)	– (0/12)	– (0/20)	– (0/10)	0.4 ± 0.01 (5/35)
	Malassezia pachydermatis (N = 21)	– (0/12)	0.31 ± 0.01 (19/20)	– (0/10)	0.38 ± 0.02 (2/35)
	Total	0.35 ± 0.02	0.35 ± 0.02	0.32 ± 0.02	0.39 ± 0.02
Keratinase activity Mean (Pz) ± SD	Malassezia globosa (N = 20)	0.75 ± 0.02 (5/12)	– (0/20)	0.62 ± 0.02 (8/10)	0.85 ± 0.02 (7/35)
	Malassezia restricta (N = 10)	1 (1/12)	– (0/20)	0.65 ± 0.02 (1/10)	1 (8/35)
	Malassezia furfur (N = 14) Malassezia sympodialis (N = 7)	0.82 ± 0.02 (6/12) – (0/12)	0.53 ± 0.02 (1/20) – (0/20)	0.65 ± 0.02 (1/10) – (0/10)	0.96 ± 0.02 (6/35) 0.84 ± 0.02 (7/35)
	Malassezia slooffiae (N = 5)	– (0/12)	– (0/20)	– (0/10)	0.8 ± 0.02 (5/35)
	Malassezia pachydermatis (N = 21)	– (0/12)	0.85 ± 0.02 (19/20)	– (0/10)	0.79 ± 0.02 (2/35)
	Total	0.86 ± 0.02	0.69 ± 0.02	0.64 ± 0.02	0.873 ± 0.02

Relative quantification of gene expression and keratinase, lipase, and phospholipase genes copy number in 20 Malassezia globosa strains_

ID	Clinical sites	RNA relative quantification			DNA relative quantification
ID	Clinical sites	Keratinase	Lipase	Phospholipase	Keratinase	Lipase	Phospholipase
S1	Folliculitis	2.11	1.84	2.59	4.43	7.22	3.06
S2	Folliculitis	2.52	7.51	7.57	1.36	2.04	1.08
S3	Folliculitis	3.52	12.99	12.40	1.50	2.12	3.36
S4	Folliculitis	0.81	3.20	3.91	0.32	12.98	0.69
S5	Folliculitis	3.40	5.55	1.48	6.58	9.57	6.36
S6	Pityriasis versicolor	0.49	0.31	0.78	0.29	0.21	0.02
S7	Pityriasis versicolor	2.65	3.52	3.17	1.35	1.46	1.10
S8	Pityriasis versicolor	2.82	2.35	2.54	2.79	7.43	1.34
S9	Pityriasis versicolor	1.25	3.10	2.53	0.77	1.60	0.24
S10	Pityriasis versicolor	2.57	2.55	2.52	1.96	1.56	3.33
S11	Pityriasis versicolor	2.84	2.75	1.32	0.97	1.74	1.03
S12	Pityriasis versicolor	0.96	2.86	3.37	0.46	0.86	2.56
S13	Pityriasis versicolor	2.55	2.86	3.38	1.50	2.58	1.88
S14	Control group	1.53	0.53	0.56	1.42	0.82	0.75
S15	Control group	1.04	1.19	2.55	0.92	1.05	0.86
S16	Control group	0.67	1.36	0.48	0.50	0.61	0.32
S17	Control group	0.94	2.37	2.43	0.50	1.06	2.33
S18	Control group	2.36	0.07	0.48	1.87	0.26	0.49
S19	Control group	0.24	2.52	2.80	0.56	1.31	1.39
S20	Control group	2.51	3.30	1.50	1.31	1.18	0.27

Biofilm production by Malassezia species isolated from different clinical sites_

Groups	Malassezia globosa		Malassezia restricta		Malassezia furfur		Malassezia sympodialis		Malassezia slooffiae		Malassezia pachydermatis		Total
Groups		Mean Abs ±SD	Range	Mean Abs ±SD	Range	Mean Abs ±SD	Range	Mean Abs ±SD	Range	Mean Abs ±SD	Range	Mean Abs ±SD	Range	Mean Abs ±SD
Folliculitis (N= 12)	0.321 ±0.01	0.155-0.41	0.366 ±0.01	0.301-0.398	0.339 ±0.01	0.317-0.594	0		0		0		0.342±0.01
Neonates (N = 20) group	0	0		0.433 ±0.02		0		0		0.361 ±0.02	0.30-0.54	0.396 ±0.02
Pityriasis versicolor (N = 10)	0.317±0.01	0.16-0.604	0.306 ±0.02		0.316 ±0.02		0		0		0		0.313 ±0.02
Control (N = 35) group	0.328±0.01	0.18-0.51	0.338±0.02	0.12-0.311	0.255 ±0.02	0.12-0.311	0.235±0.02	0.137-0.361	0.256±0.02	0.167-0.331	0.336 ±0.02	0.22-0.44	0.291 ±0.02
Total	0.322 ±0.01	0.155-0.604	0.336 ±0.02	0.12-0.572	0.336 ±0.02	0.12-0.594	0.235±0.02	0.137-0.362	0.256 ±0.03	0.167-0.332	0.349 ±0.02	0.22-0.54

Production and Quantification of Virulence Factors in Malassezia Species

Figures & Tables

Fig. 1

Fig. 2

The sequences of primers and probes used in RT-qPCR for gene expression studies in 20 Malassezia globosa isolates_

Level of phospholipase, lipase, and keratinase production by Malassezia species_

Enzymatic activities of Malassezia species isolated from different clinical sites_

Relative quantification of gene expression and keratinase, lipase, and phospholipase genes copy number in 20 Malassezia globosa strains_

Biofilm production by Malassezia species isolated from different clinical sites_

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