Production and Quantification of Virulence Factors in Malassezia Species

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

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

Table II

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

[i] N - number of tested isolates; SD - 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.

Table III

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)

[i] N - number of tested isolates

n - number of isolates with positive activity for the corresponding hydrolytic enzyme

SD - standard deviation

Table IV

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

[i] N– number of tested isolates; SD – standard deviation

Table V

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

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DOI: https://doi.org/10.33073/pjm-2022-047 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331

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Language: English

Page range: 529 - 538

Submitted on: Jul 12, 2022

Accepted on: Sep 20, 2022

Published on: Dec 6, 2022

Published by: Polish Society of Microbiologists

In partnership with: Paradigm Publishing Services

Publication frequency: 4 issues per year

Keywords:

Malassezia,

Real-Time qRT-PCR,

virulence factors,

extracellular hydrolases,

biofilm

Related subjects:

Life sciences,

Microbiology and virology

© 2022 Inès Hadrich, Nahed Khemekhem, Sourour Neji, Houaida Trablesi, Amin Ilahi, Hayet Sellami, Fattouma Makni, Ali Ayadi, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Volume 71 (2022): Issue 4 (December 2022)

Production and Quantification of Virulence Factors in Malassezia Species

Figures & Tables

Table I

Fig. 1

Table II

Fig. 2

Table III

Table IV

Table V

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